Plant Cell Advance Publication. Published on October 14, 2016, doi:10.1105/tpc.16.00295
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LARGE-SCALE BIOLOGY ARTICLE
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Global Analysis of Truncated RNA Ends Reveals New Insights
into Ribosome Stalling in Plants
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Cheng‐Yu Houa, Wen‐Chi Leea, Hsiao‐Chun Choua, b, Ai‐Ping Chenc, Shu‐Jen
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Chouc and Ho‐Ming Chena1
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Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan
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Institute of Plant Biology, National Taiwan University, Taipei, 10617, Taiwan
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Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan
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Address correspondence to [email protected]
Short title: Ribosome-Protected Fragments in the RNA Degradome
The author responsible for distribution of materials integral to the findings presented in
this article in accordance with the policy described in the Instructions for Authors
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(www.plantcell.org) is: Ho-Ming Chen ([email protected]).
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One-sentence summary: In a global analysis of free 5' mRNA ends, this study shows
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that the plant RNA degradome contains in vivo ribosome footprints and is useful to
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study ribosome stalling during translation.
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ABSTRACT
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cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA
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(Arabidopsis thaliana), rice (Oryza sativa) and soybean (Glycine max). Based on this
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frame. By examining conserved peptide upstream open reading frames (uORFs) of
High-throughput approaches for profiling the 5′ ends of RNA degradation
intermediates on a genome-wide scale are frequently applied to analyze and validate
degradome other than miRNA targets is currently largely uncharacterized, and this
limits the application of RNA degradome studies. We conducted a global analysis of
5′-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis
analysis, we provide multiple lines of evidence to show that the plant RNA degradome
contains in vivo ribosome-protected mRNA fragments. We
observed a 3-nucleotide
(nt) periodicity in the position of free 5' RNA ends and a bias toward the translational
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©2016 American Society of Plant Biologists. All Rights Reserved
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Arabidopsis and rice, we found a predominance of 5′ termini of RNA degradation
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intermediates that were separated by a length equal to a ribosome-protected mRNA
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Arabidopsis ARGONAUTE7 to a non-cleavable target site of miR390 might directly
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INTRODUCTION
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Steady-state levels of RNA are controlled by relative rates of transcription and
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RNA degradation. Most mRNAs in eukaryotes possess a 7-methylguanosine
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cap at the 5′ terminus and a poly(A) tail at the 3′ terminus which are crucial for
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translation and RNA stability. The loss of the 5′ cap or the 3′ poly(A) tail
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abolishes mRNA translation and promotes mRNA degradation (Gallie, 1991).
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Uncapped 5′ ends of mRNAs are degraded by 5′-3′ exoribonucleases (XRNs)
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whereas deadenylated mRNAs are degraded by the exosome from the 3′ end
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(Lebreton and Seraphin, 2008; Houseley and Tollervey, 2009). Alternatively,
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deadenylation can also trigger decapping of mRNA, followed by degradation
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from the 5′ end (Muhlrad et al., 1994).
fragment. Through the analysis of RNA degradome data, we discovered uORFs and
CDS regions potentially associated with stacked ribosomes in Arabidopsis.
Furthermore, our analysis of RNA degradome data suggested that the binding of
hinder ribosome movement. This work demonstrates an alternative use of RNA
degradome data in the study of ribosome stalling.
Translation plays a crucial role in controlling mRNA stability and is required
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to
eliminate
aberrant
mRNAs
through
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(Shoemaker and Green, 2012). Nonsense-mediated mRNA decay (NMD) is
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often initiated when a ribosome encounters a premature termination codon
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(PTC) upstream of an exon junction complex (EJC). The interaction between
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NMD factors on termination factors and EJCs promotes the degradation of
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PTC-containing transcripts. The movement of ribosomes on mRNA molecules
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can be stopped externally by a stable RNA structure or internally by the
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particular peptide that is encoded by the mRNA. The transcripts associated
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several
distinct
mechanisms
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with stalled ribosomes are degraded by a specialized RNA surveillance
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pathway called no-go decay, which may result in endonucleolytic cleavage
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upstream of the stalled ribosomes (Doma and Parker, 2006). Non-stop decay
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also targets transcripts with stalled ribosomes, but the stalling is due to the lack
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of an in-frame stop codon (Frischmeyer et al., 2002; van Hoof et al., 2002).
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Many eukaryotic mRNAs possess short open reading frames (ORFs) in the 5′
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untranslated region (UTR) which can be translated and potentially regulate
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mRNA stability and translation. The stop codon of a translated upstream open
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reading frame (uORF) might be recognized as a PTC and thus initiate NMD.
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Some uORFs encoding peptides conserved across species have been shown
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to stall ribosomes at uORF stop codons, resulting in the repression of
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downstream main ORF translation and acceleration of RNA degradation in a
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few cases (Gaba et al., 2005; Uchiyama-Kadokura et al., 2014).
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In eukaryotes, small RNAs of 20 to 30 nt play a key role in regulating gene
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expression through the RNA interference pathway. Most animal microRNAs
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(miRNAs) have a seed region, spanning the second to the seventh or the
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eighth nucleotide, which can base pair perfectly with the 3′ UTR of a target
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mRNA (Bartel, 2009). The targeting of animal miRNAs is often associated with
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translation
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exoribonucleases. By contrast, plant miRNAs are highly complementary to
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their targets, and cleavage in the middle of target sites is the major mode of
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plant miRNA action (Rhoades et al., 2002). However, growing evidence
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indicates that plant miRNAs can also inhibit the translation of their targets
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(Brodersen et al., 2008; Iwakawa and Tomari, 2013; Li et al., 2013b; Li et al.,
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2013c; Liu et al., 2013). In vitro assays of RNA-induced silencing complexes
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(RISCs) containing Arabidopsis (Arabidopsis thaliana) ARGONAUTE1 (AGO1)
repression,
deadenylation
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and
mRNA
decay
using
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demonstrated that plant miRNAs can repress translation initiation uncoupled
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with deadenylation or mRNA decay (Iwakawa and Tomari, 2013). Furthermore,
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miRNA binding sites in ORFs can hinder the movement of ribosomes
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(Iwakawa and Tomari, 2013). In Arabidopsis, miRNA-mediated translation
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repression occurs in the endoplasmic reticulum and requires the endoplasmic
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reticulum protein ALTERED MERISTEM PROGRAM1 (Li et al., 2013c).
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High-throughput approaches for genome-wide profiling of RNA degradation
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intermediates that possess a free monophosphate at the 5′ terminus have
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been developed by several groups and are variously named parallel analysis
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of RNA ends (PARE) (German et al., 2008), degradome sequencing
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(Addo-Quaye et al., 2008), genome-wide mapping of uncapped transcripts
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(GMUCT) (Gregory et al., 2008), and 5′ P sequencing (5Pseq) (Pelechano et
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al., 2015). Because intact mRNAs generally possess a 5′ cap that blocks their
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ligation to RNA adaptors, truncated 5′ RNA ends with a free monophosphate
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can be selectively sequenced by directly ligating poly(A) RNA with RNA
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adaptors. PARE and degradome sequencing have been widely applied in the
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identification of small RNA-guided cleavage sites in various plant species
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(Addo-Quaye et al., 2008; German et al., 2008; Zhou et al., 2010;
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Shamimuzzaman and Vodkin, 2012; Zhao et al., 2012; Li et al., 2013a).
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Specific PARE has been developed for the study of plant miRNA processing by
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specifically amplifying miRNA processing intermediates (Bologna et al., 2013).
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Although these approaches have been used to profile mRNA degradation
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intermediates in mutants impaired in XRNs or proteins with endonucleolytic
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activity (German et al., 2008; Harigaya and Parker, 2012; Schmidt et al., 2015),
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the interpretations of some results remain challenging because the complexity
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of the RNA degradome is currently still largely uncharacterized.
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Previously, we showed that plant RNA degradome data potentially contain
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footprints of RNA binding proteins in the 3′ UTR (Hou et al., 2014). Pelechano
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et al. (2015) also demonstrated that yeast 5Pseq data contain in vivo ribosome
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footprints, the products of co-translation mRNA decay. The change in these
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ribosome-protected termini captured by 5Pseq could reflect ribosome
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dynamics without the problems caused by translational inhibitors sometimes
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used in the generation of in vitro ribosome footprints. Codons associated with
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paused ribosomes in response to oxidative stress were identified from the
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analysis of yeast 5Pseq data. Co-translation mRNA decay was also
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demonstrated to be mediated by XRN4 and involved in the reprogramming of
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gene expression under heat stress in Arabidopsis (Merret et al., 2013; Merret
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et al., 2015). Here, we further demonstrate that footprints of ribosomes are
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widespread in the RNA degradome of various plant species. Our genome-wide
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analysis of RNA degradation fragments revealed uORFs, CDS regions and
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non-cleavable miRNA target sites that are potentially associated with stacked
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ribosomes. Our findings thus expand the application of plant RNA degradome
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data for the elucidation of post-transcriptional gene regulation beyond small
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RNA-guided cleavage.
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RESULTS
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Signatures of Ribosome Footprints Were Observed in the RNA
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Degradome
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Our previous analysis of plant RNA degradome data revealed positional
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enrichment of 5′-truncated RNA ends in the proximity of motifs recognized by
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RNA-binding proteins. This suggests that RNA-binding proteins attached to
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RNA may hinder RNA degradation and result in protected RNA fragments (Hou
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et al., 2014). Therefore, we suspected that the binding of ribosomes to mRNA
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may also protect mRNA from in vivo degradation and leave ribosome footprints
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in the RNA degradome. To explore this possibility, we first used PARE data to
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plot the positional distribution of 5′-truncated mRNA ends in the junctions of the
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CDSs and UTRs. We predicted that, if PARE can capture ribosome footprints
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in the same way as ribosome profiling (Ribo-Seq), which delineates ribosome
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positions by generating ribosome-protected mRNA fragments through in vitro
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nuclease digestion (Ingolia, 2010), we would observe a 3-nt periodicity in the
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CDSs. A 3-nt periodicity reflects the stepwise movement of ribosomes during
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active translation and has been reported in the analyses of Ribo-Seq data
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derived from multiple species (Ingolia et al., 2009; Guo et al., 2010; Ingolia et
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al., 2011; Liu et al., 2013; Bazzini et al., 2014; Juntawong et al., 2014; Vasquez
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et al., 2014). Consistent with our prediction, 5′ ends of truncated mRNA (PARE
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data) generated from Arabidopsis seedlings and inflorescences show strong
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3-nt phasing in the 3′ terminus of the CDSs but not in the proximal region of the
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3′ UTR (Figure 1A). A 3-nt periodicity in the CDS region was also observed in
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the CDSs for PARE reads generated from rice (Oryza sativa) inflorescences
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and soybean (Glycine max) seeds (Figure 1A). Besides this phasing pattern,
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the three species all show preferential accumulation of PARE reads in the
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translational frame (frame 1) of annotated CDSs (Figure 1B). Although the
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enrichment in the translational frame is relatively small in PARE data
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compared to that in yeast Ribo-Seq data reported previously (Ingolia et al.,
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2009), the proportion of PARE reads falling in the translational frame is
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significantly higher than that in the other two frames in the three replicates of
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Arabidopsis inflorescence PARE data (Supplemental Figure 1). Similar to the
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previous finding in the analysis of Ribo-Seq data (Liu et al., 2013; Bazzini et al.,
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2014; Juntawong et al., 2014), PARE data of these three plant species also
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showed an evident increase in the number of reads at positions 16 and 17 nt
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upstream of stop codons, a pattern consistent with the deceleration of
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ribosome movement during translational termination (Figure 1A). These
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common features shared between PARE data and Ribo-Seq data strongly
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suggest both the presence of in vivo ribosome-protected mRNA fragments in
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the plant RNA degradome and the occurrence of co-translational RNA decay in
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plants.
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Regular and Conserved RNA Degradation Patterns Were Found in
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Conserved Peptide uORFs
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Several uORFs in fungi, plants, and animals that encode conserved peptides
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are able to block ribosomes at stop codons (Wang and Sachs, 1997; Raney et
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al., 2000; Gaba et al., 2001; Hayden and Jorgensen, 2007; Hood et al., 2007;
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Uchiyama-Kadokura et al., 2014). Among them, the conserved peptide uORFs
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(CPuORFs)
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S-ADENOSYLMETHIONINE DECARBOXYLASE (SAMDC/AdoMetDC1) have
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been demonstrated to induce mRNA decay through the NMD pathway (Gaba
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et al., 2005; Uchiyama-Kadokura et al., 2014). To provide additional evidence
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that the RNA degradome contains ribosome footprints, we examined the
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positional distribution of 5′-truncated mRNA ends (PARE reads) derived from
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several CPuORFs of Arabidopsis. In SAMDC, the position 16 nt upstream of
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the uORF stop codon shows a predominant accumulation of PARE reads
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derived from seedlings but only a weak enrichment of PARE reads derived
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from inflorescences (Supplemental Figure 2). Besides SAMDC, we also
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examined PARE reads that mapped to the CPuORFs in a small group of bZIP
of
yeast
CPA1
and
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an
Arabidopsis
gene
producing
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genes which regulate the translation of downstream ORFs in response to
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sucrose concentration (Wiese et al., 2004). Interestingly, these bZIP CPuORFs
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possess a ladder of PARE peaks at intervals of about 30 nt, which is the size of
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a ribosome-protected fragment in Arabidopsis (Liu et al., 2013; Juntawong et
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al., 2014) (Figure 2A). Counting from the 3′ end of bZIP CPuORFs, the first
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and second PARE peaks are positioned about 16 and 46 nt upstream of uORF
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stop codons, with a few reads present in the 30-nt window between these two
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peaks. A third PARE peak at position -76 that was an additional 30-nt upstream
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was observed in bZIP2 and bZIP11 CPuORFs (Figure 2A). The 30-nt phasing
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of PARE peaks in the 3′ end of CPuORFs provides strong evidence to support
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the notion that PARE captures the degradation fragments protected by an
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array of stacked ribosomes. These three PARE peaks likely delineate the 5′
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ends of degradation fragments protected by one, two, or three adjacent
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ribosomes stalled at a CPuORF stop codon, respectively. Notably, the 30-nt
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phasing was not evident in these bZIP genes when we analyzed in vitro
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ribosome-protected mRNA fragments of two Ribo-Seq data sets that were
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generated by independent groups (Liu et al., 2013; Juntawong et al., 2014)
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(Figure 2A; Supplemental Figure 3).
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Because bZIP CPuORFs were reported to repress the translation of the
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downstream ORF under high sucrose concentration (Wiese et al., 2004), we
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used modified RNA ligase-mediated rapid amplification of cDNA ends (RLM 5′
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RACE) to test whether treatment with 6% sucrose would affect the
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accumulation of degradation fragments truncated at these bZIP CPuORFs.
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Indeed, a larger number of 5′ ends of degradation intermediates corresponding
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to the first or second peak in bZIP2 and bZIP11 CPuORFs were detected in
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Arabidopsis seedlings treated with 6% sucrose compared to the untreated
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control plants (Figure 2B). This implies either an increase in ribosomes
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arrested at these two uORFs or the enhancement of RNA degradation. Taken
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together, these results indicate that degradation fragments appear to reflect
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the dynamics of ribosomes on uORFs.
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Although 64 CPuORFs (Hayden and Jorgensen, 2007) have been identified
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in Arabidopsis by sequence comparison and annotated in The Arabidopsis
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Information Resource database (TAIR10 annotation), most of them have not
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been demonstrated to arrest ribosomes at specific positions. To further explore
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whether ribosome stalling at stop codons is a common mechanism underlying
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the regulation mediated by Arabidopsis CPuORFs, we globally analyzed the
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distribution of PARE reads mapped to the 3′ end of the 64 Arabidopsis
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CPuORFs. Some Arabidopsis CPuORFs predominantly accumulate PARE
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reads at position 16 nt upstream of stop codons and some show an additional
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peak at position -45, -46 or -47 (Figure 3A). Besides bZIP genes, CPuORFs in
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genes encoding several basic helix-loop-helix type transcription factors, a
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trehalose-6-phosphate phosphatase, two methyltransferases and an unknown
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protein were found to possess PARE peaks at these two specific sites
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(Supplemental Figure 4), implying the stalling of ribosomes at these uORF
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stop codons. The analysis of 35 rice CPuORFs showed the enrichment of
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PARE reads at the same sites (positions -16 and -46) (Figure 3A), suggesting
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that ribosome stalling at the stop codon of some CPuORFs is a conserved
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mechanism across species. To know whether ribosome profiling could capture
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the signature of ribosome stacking in CPuORFs as PARE, we also analyzed in
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vitro ribosome-protected mRNA fragments of two Ribo-Seq data sets with the
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number of total reads greatly exceeding that of PARE data sets we used (Liu et
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al., 2013; Juntawong et al., 2014). Intriguingly, although position 16 or 17 nt
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upstream of the uORF stop codon shows an enrichment of in vitro
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ribosome-protected mRNA fragments, the enrichment at these positions is less
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prominent and does not accompany the enrichment at the positions near 30 nt
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upstream (Figure 3B). The same analysis on genome-wide predicted uORFs
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having lengths greater than 60 nt showed no preferential accumulation of
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PARE reads for Arabidopsis or rice and Arabidopsis Ribo-Seq reads if
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predicted uORFs overlapping CPuORFs were excluded (Figures 3A and 3B).
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Based on the analysis of PARE data which harbor ribosome footprints, most
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Arabidopsis and rice uORFs may not cause ribosome stalling at stop codons in
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the same way as many CPuORFs, at least under the conditions and in the
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tissues that the PARE data were generated.
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Regulatory uORFs Were Identified Using the Patterns of RNA
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Degradation Fragments
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We suspected that a few regulatory uORFs may not be conserved between
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Arabidopsis and rice or that they might have been missed in the previous
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search because of low sequence homology (Hayden and Jorgensen, 2007).
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Therefore, analysis of the RNA degradome might provide an alternative
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approach for the identification of regulatory uORFs that have the potential to
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stall ribosomes. We thus reverse searched for uORFs using PARE reads
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peaking at the regions 16-17 and 45-47 nt upstream of the stop codon of
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predicted uORFs. In addition to four CPuORFs reported previously (Hayden
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and Jorgensen, 2007), we identified four Arabidopsis uORFs with two
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predominant PARE peaks representing two tandem ribosomes stacking at stop
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codons (Table 1 and Supplemental Figure 5). Further analyses were then
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performed on these three novel uORFs to investigate sequence conservation
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and their regulatory functions.
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A 99-nt uORF in the 5′ UTR of CBL-INTERACTING PROTEIN KINASE 6
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(CIPK6) shows two PARE peaks at positions 16 and 46 nt upstream of the stop
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codon like CPuORFs in bZIP genes (Figures 2 and 4A). We then compared the
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distribution of reads obtained through Ribo-Seq and PARE on CIPK6. The
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Ribo-Seq data of light-treated seedlings show a corresponding peak at
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position 16 nt but not 46 nt upstream of the stop codon (Figure 4A). Moreover,
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position -17 accumulates more reads than position -16 in Ribo-Seq data,
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whereas position -16 has the highest accumulation of PARE reads.
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Predominant accumulation of ribosome-protected 5′ ends at positions 16 and
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46 upstream of CIPK6 stop codon was not observed in the Ribo-Seq data of
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normoxia (in air) seedlings (Supplemental Figure 6). Although the CIPK6
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uORF was not annotated in the TAIR database, it encodes a conserved
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peptide and has been identified previously based on sequence homology by
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two groups (Takahashi et al., 2012; Vaughn et al., 2012). Similarly, the
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conserved uORF in soybean CIPK6 also possesses PARE peaks in 30-nt
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increments at the same positions relative to the uORF stop codon (Figure 4B),
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implying that ribosome stalling might be a conserved mechanism for CIPK6
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uORF regulation.
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The function of Arabidopsis CIPK6 uORF in repressing downstream ORF
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expression was validated via transient expression assays by Ebina et al.
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(2015). We confirmed the regulatory function of Arabidopsis CIPK6 uORF by
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generating stable transgenic lines which harbored a reporter gene encoding
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β-glucuronidase (GUS) driven by the Arabidopsis CIPK6 promoter containing a
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wild-type (WT) 5' UTR or a uORF-deleted (ΔuORF) 5' UTR in which the start
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codon was converted into a stop codon (Figure 4C). Overall, the ΔuORF
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transgenic lines showed a higher level of expression of the reporter gene in
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most tissues (Figure 4C; Supplemental Figure 7), indicating that the regulation
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mediated by CIPK6 uORF is widespread even under normal growth conditions.
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Because an approximate 6-fold difference (P = 0.02, two-tailed student’s t-test)
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was detected in the comparison of GUS activity, but the change of GUS mRNA
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level was less than 2-fold and not statistically significant (Figures 4D and 4E),
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CIPK6 uORF likely controls downstream ORF expression mainly at the
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translational level.
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The other two candidates of ribosome stalling uORFs we identified are
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located in MYB34 and MYB51, which belong to the same clade of MYB
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transcription factors involved in the regulation of glucosinolate biosynthesis
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(Celenza et al., 2005; Gigolashvili et al., 2007; Frerigmann and Gigolashvili,
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2014). Although MYB34 uORF is longer than MYB51 uORF, they both possess
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two predominant PARE peaks at positions 16 and 46 nt upstream of uORF
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stop codons (Figure 5A). The translation of these two MYB uORFs is
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supported by the higher density of Ribo-Seq reads in the predicted ORFs
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compared to that in the flanking regions (Liu et al., 2013) (Figure 5A). However,
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the analysis of in vitro ribosome-protected mRNA fragments on these two MYB
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uORFs with two Ribo-Seq data sets (Liu et al., 2013; Juntawong et al., 2014)
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showed no preferential accumulation at these two sites (Figure 5A;
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Supplemental Figure 6). The peptide sequences encoded by these two uORFs
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are highly conserved at the C-terminus within the mustard (Brassicaceae)
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family (Figure 5B). Glucosinolates are sulfur- and nitrogen-containing
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secondary metabolites that are found mainly in plant species in the order of
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Brassicales (Grubb and Abel, 2006), explaining the absence of conserved
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uORFs in many other plant species. The negative impact of MYB34 CPuORF
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on the mRNA level of MYB34 was demonstrated previously in a study of a
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mutant containing a PTC in this uORF (Bender and Fink, 1998). We confirmed
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the negative regulation of MYB34 uORF on the expression of the downstream
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reporter gene by converting the start codon into a stop codon in transient
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expression assays (Figure 5C). The abolition of MYB51 uORF slightly
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increased the expression of the reporter gene, but the change was not
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statistically significant under the conditions we used for transient expression
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assays (Figure 5C). The identification of MYB34 and MYB51 uORFs validates
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the use of RNA degradome data in discovering lineage or species specific
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regulatory uORFs.
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Ribosome Stacking Was Predicted in CDSs Using the RNA Degradome
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Data
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Besides CPuORFs, the nascent peptide encoded by a CDS region in the first
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exon of Arabidopsis CGS1, known as the MTO1 region, has been reported to
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block ribosome elongation and induce RNA decay in response to
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S-adenosyl-L-methionine (AdoMet) (Onouchi et al., 2005). Similar to the
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degradation fragments we observed in bZIP CPuORFs (Figure 2), Haraguchi
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et al. (2008) detected a ladder of truncated 5′ termini separated in length by
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approximately 30-nt increments in the MTO1 region together with the 5′
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upstream region after treatment with AdoMet. Moreover, the previous study
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demonstrated that the truncated mRNA ends are defined by the 5′ edges of
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stalled ribosomes in an array. Therefore, we assumed that the RNA
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degradome could also be used in the identification of ribosome stacking
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occurring in the CDS region. To validate this idea, we first compared the PARE
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peaks around the MTO1 region with the 5′ ends of degradation intermediates
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reported previously (Yamashita et al., 2014). Although the plants we used for
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PARE library construction were grown in soil without AdoMet treatment, the
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major PARE peaks were in close proximity to the 5′ termini of degradation
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intermediates reported previously (Supplemental Figure 8).
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Next, we performed phasing analysis on PARE peaks in the CDSs for
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intervals from 20 to 40 nt. In the analysis of Arabidopsis seedling PARE data,
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the number of phased regions decreased when the length of intervals
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increased (Supplemental Figure 9A). However, the numbers of phased regions
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identified at intervals of 32 to 40 nt and 28 nt were significantly lower than that
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at the interval of 30 nt in the inflorescence PARE data (Supplemental Figure
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9B). To eliminate the phased regions occurring by chance, we discarded
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candidates identified only in a single inflorescence sample. This resulted in
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four phased regions at the intervals of 29 and 30 nt and one to three phased
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regions at the other intervals (Supplemental Figure 9C). Based on this result,
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we thus discovered four Arabidopsis protein-coding genes possessing a region
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potentially associated with protection signatures of stacked ribosomes (Figure
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6). PARE peaks with prominent phasing were identified in genes encoding
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plastidic type I signal peptidase 2B (PLSP2B), a pentatricopeptide repeat
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protein, a RING/U-box superfamily protein, and an unknown protein (Figure 6).
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However, Ribo-Seq peaks separated in a 30-nt interval were not detected in
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these regions, although the position of the most 3′ phased PARE peak
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identified in PLSP2B showed predominant accumulation of Ribo-Seq reads
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(Liu et al., 2013; Juntawong et al., 2014) (Figure 6; Supplemental Figure 10).
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Notably, the A sites of stalled ribosomes putatively associated with these four
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regions were all predicted to fall in frame 2 but not in the translational frame
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annotated in TAIR (frame 1) if we assumed that the distance from the
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ribosome-protected 5′ end to the presumed A site was 17 nt according to the
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result shown in Figure 1. Stalling in the non-translational frame may suggest
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that the last pausing ribosome in the four CDS regions we identified is arrested
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during the step of translocation but not decoding.
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Analysis of 5′-Truncated mRNA Ends Upstream of miRNA-Guided
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Cleavage Sites
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Plant miRNAs can guide cleavage in the middle of target sites, resulting in
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truncated mRNA fragments (Rhoades et al., 2002). However, growing
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evidence suggests that plant miRNA can repress target translation (Brodersen
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et al., 2008; Iwakawa and Tomari, 2013; Li et al., 2013b; Li et al., 2013c; Liu et
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al., 2013), although the underlying mechanism is not well characterized.
391
Because our analysis of the RNA degradome revealed the footprints of stalled
392
ribosomes in uORFs and the CDSs, we predicted that a similar analysis of
393
miRNA target genes would be useful for elucidating whether the binding of
394
plant miRNAs can directly block the movement of ribosomes in planta.
395
Therefore, we investigated the distribution of PARE reads in a 55-nt region
396
upstream of putative miRNA-guided cleavage sites in Arabidopsis and rice.
397
Unlike the predominant accumulation of PARE reads at positions 16 and 46 nt
398
upstream of uORF stop codons (Figure 2), no position-specific enrichment was
399
found in this region except at the putative miRNA-guided cleavage sites
400
(Figure 7). This result thus suggests that the major mechanism by which plant
401
miRNAs repress target translation may not be by acting as physical barriers to
402
hinder the movement of ribosomes.
403
404
Discovery of Potential Footprints of Ribosomes Hindered by AGO7 in
15
405
Arabidopsis TAS3
406
Surprisingly, three Arabidopsis TAS3 genes shared a highly similar but unusual
407
pattern of truncated 5′ RNA ends upstream of the non-cleavable target site of
408
miR390 (Figure 8A). A PARE peak was located immediately or 4 nt upstream
409
of the first base of non-cleavable target sites of miR390. In the proximal region
410
upstream of this peak, there were three additional PARE peaks arranged at
411
regular intervals of 28 to 30 nt. This regular degradation pattern in TAS3 highly
412
resembled that detected in bZIP CPuORFs (Figure 2). Therefore, we assumed
413
that the peak adjacent to the non-cleavable miRNA site might be the footprint
414
of AGO7, which loads miR390, whereas the upstream peaks likely also
415
represented ribosome footprints. Intriguingly, TAS3 was annotated to be
416
non-coding RNA because of lack of a long ORF and the production of
417
conserved trans-acting siRNAs (tasiRNAs) when targeted by miR390 (Allen et
418
al., 2005).
419
To determine whether TAS3 might serve as a template for translation, we
420
first predicted ORFs in three Arabidopsis TAS3 genes. All three TAS3 genes
421
have an ORF that terminates 7 to 11 nt upstream of the non-cleavable site of
422
miR390 (Figure 8A). The coding nature of the predicted ORFs in three TAS3
423
genes was evaluated with Ribo-Seq data published previously (Liu et al., 2013;
424
Juntawong et al., 2014). The predicted ORFs in TAS3a and TAS3b have
425
relatively dense ribosome-protected fragments compared to other regions,
426
supporting the coding ability (Figure 8A; Supplemental Figure 11). Moreover,
427
the major peaks revealed by Ribo-Seq were close to the predominant PARE
428
peaks in TAS3a and TAS3b. This result suggested that translated ORFs were
429
present upstream of the non-cleavable target site of miR390 and 5′-truncated
430
RNA ends mapped to this region were likely ribosome footprints. If these PARE
16
431
peaks represent the footprints of ribosomes hindered by miR390-AGO7
432
complex but not the cleavage remnants directed by tasiRNAs, they should
433
disappear in the ago7 but be sustained in the mutant of RNA-DEPENDENT
434
RNA POLYMERASE 6 (RDR6), which acts downstream of AGO7 in the
435
tasiRNA biogenesis pathway (Mallory and Vaucheret, 2010). As predicted, the
436
result of modified RLM 5′ RACE clearly showed that degradation fragments
437
with 5′ ends mapped to these positions were barely detected in ago7 (Figure
438
8B). On the other hand, the amounts of these degradation fragments were
439
comparable between WT and rdr6 plants although both ago7 and rdr6 showed
440
increased amounts of TAS3a transcripts compared to the WT and no tasiRNA
441
production (Figures 8B, 8C, and 8D). The PARE data of WT, rdr6 and ago7
442
inflorescences showed the same result as the modified RLM 5′ RACE assay.
443
All PARE peaks in the ORFs of three TAS3 genes vanished in ago7 except the
444
one at position -58 in TAS3b (Supplemental Figure 12). The result of mutant
445
analysis, therefore, indicates that the formation of these 5′-truncated RNA ends
446
upstream of the non-cleavable target site of miR390 depends on AGO7 but is
447
independent
448
non-cleavable targeting of AGO7 may arrest ribosomes as a road block in
449
planta. Because the last major PARE peaks in the ORFs in TAS3a, TAS3b and
450
TAS3c are located 19, 14 and 16 nt upstream of the first nucleotide of the stop
451
codons respectively, the miR390-AGO7 complex appears to arrest a ribosome
452
in the step of elongation on TAS3a but in the step of termination on TAS3b and
453
TAS3c.
of
tasiRNA.
Taken
together,
these
results
imply
that
454
455
DISCUSSION
456
The
RNA
degradome
is
composed
17
of
degradation
products
of
457
endoribonucleases and XRNs acting through diverse pathways. Here, we not
458
only demonstrate that ribosome-protected mRNA ends are pervasive in the
459
RNA degradome,but also demonstrate exciting new applications of RNA
460
degradome data in the study of post-transcriptional gene regulation.
461
Predominant accumulation of 5′-truncated mRNA ends in a 30-nt interval likely
462
represents an array of stacked ribosomes in the uORF, CDS, or the upstream
463
region of non-cleavable binding sites of AGO7 (Figure 9). Genome-wide
464
analysis of 5′ -truncated mRNA ends mapped to uORFs suggests that many
465
CPuORFs may repress downstream ORF expression by stalling ribosomes at
466
CPuORF stop codons, whereas the majority of predicted uORFs lack this
467
ability (Figure 3). In addition, the analysis of 5′-truncated mRNA ends occurring
468
upstream of miRNA target sites uncovered the signature of ribosome stacking
469
in the non-cleavable target site of miR390 but not other cleavable target sites
470
(Figures 7 and 8). Novel regulatory uORFs and CDS regions with potential to
471
cause ribosome stalling were identified through the analysis of the RNA
472
degradome (Figures 4, 5, 6 and Table 1).
473
474
Comparison of Ribosome Footprints in RNA Degradome Data and
475
Ribo-Seq Data
476
Accumulation of degradation intermediates starting at the 5′ edge or the A site
477
of stalled ribosomes has been reported in bacteria, yeast, and Arabidopsis
478
(Hayes and Sauer, 2003; Sunohara et al., 2004; Doma and Parker, 2006;
479
Haraguchi et al., 2008), and it has been proposed that endoribonucleases are
480
involved in the production of these degradation fragments. However, a recent
481
genome-wide survey of monophosphorylated 5′ RNA ends in yeast showed
482
that XRN1 is also involved in the accumulation of 5′-truncated RNA ends
18
483
protected by ribosomes during RNA decay (Pelechano et al., 2015). Using the
484
plant degradome data, we identified an array of 5′-truncated RNA ends
485
separated at 30-nt intervals in uORFs and CDSs, implying an array of stacked
486
ribosomes (Figures 2, 4, 5, and 6). However, the 30-nt phasing pattern was not
487
observed in Ribo-Seq data for the regions we identified from RNA degradome
488
analysis. The discrepancy between Ribo-Seq data and RNA degradome data
489
is likely due to the different procedures of library preparation, although both
490
methods could capture ribosome-protected mRNA fragments. A typical
491
Ribo-Seq protocol includes a step to select fragments protected by a single
492
ribosome but not stacked disomes or trisomes (Ingolia, 2010). Therefore, a
493
region associated with stacked ribosomes may rather show a low density of
494
protected fragments in Ribo-Seq data. On the other hand, degradome
495
sequencing, PARE, GMUCT or 5Pseq profiles truncated RNA termini without
496
monosome selection and can thus capture fragments protected by stacked
497
ribosomes. In addition, in the absence of monosome selection and translation
498
inhibitor treatment to block ribosomes on mRNA, these methods may also
499
allow the identification of fragments protected by ribosomes on distinct
500
translation states.
501
A previous global study of yeast ribosome footprints demonstrated that
502
ribosome-protected fragments fall into two major groups (28-30 nt long and
503
20-22 nt long) that are stabilized by different translation inhibitors (Lareau et al.,
504
2014). The size of the protected fragments reflects the configuration and
505
translation state of the ribosomes. In the three Arabidopsis TAS3 genes, we
506
detected PARE peaks at 24-30 nt immediately upstream of the first base of
507
non-cleavable miR390 target sites (Figure 8A). The shorter intervals between
508
two PARE peaks may imply a ribosome pausing at the translocation stage
19
509
instead of the decoding stage, which should lead to a 30-nt protected fragment
510
in Arabidopsis. In addition to technical differences, the interplay between
511
ribosome stalling and RNA decay likely enhances the signal of stalled
512
ribosomes in RNA degradome data. By contrast, the signal of stalled
513
ribosomes might be embedded in the footprints of active ribosomes in
514
Ribo-Seq data. In summary, the use of degradome data for the identification of
515
regions associated with stacked ribosomes may outperform the use of
516
Ribo-Seq data in some cases.
517
518
Investigation of Regulatory uORFs using the RNA Degradome
519
Comprehensive identification of regulatory uORFs has been challenging as
520
predicted uORFs may not be translated or regulate downstream ORF
521
expression through distinct mechanisms (Barbosa et al., 2013). Prior to the
522
development of Ribo-Seq, translated uORFs with regulatory functions were
523
often identified by mutant screening or evolutionary conservation (Hill and
524
Morris, 1993; Delbecq et al., 1994; Wiese et al., 2004; Imai et al., 2006;
525
Hayden and Jorgensen, 2007). Although Ribo-Seq data have been used in the
526
identification of translated uORFs (Fritsch et al., 2012; Liu et al., 2013; Ingolia
527
et al., 2014), the application of Ribo-Seq data to the identification of ribosome
528
stalling uORFs has not been reported. In this study, we show that some
529
CPuORFs over-accumulate 5′-truncated RNA ends with a signature of stacked
530
ribosomes (Figure 2A). We further demonstrate that the accumulation of
531
5′-truncated RNA ends in bZIP CPuORFs is enhanced in response to a high
532
concentration of sucrose (Figure 2B). The data thus support the previous
533
hypothesis that the conserved peptides encoded by these bZIP CPuORFs can
534
stall ribosomes at stop codons in response to sucrose concentration (Wiese et
20
535
al., 2004), and suggest the application of degradome data in the study of
536
regulatory uORFs. Through the global analysis of 5′-truncated RNA ends
537
occurring in predicted uORFs, we identified novel ribosome stalling uORFs in
538
Arabidopsis. Notably, uORFs in MYB34 and MYB51 are conserved and
539
specific in the Brassicaceae family (Figure 5B). Since RNA degradome data of
540
diverse plant species are available in the public domain, these data sets might
541
be very useful for the identification of lineage or species specific ribosome
542
stalling uORFs as evolutionary conservation is not required for this type of
543
analysis.
544
545
Analysis of the RNA Degradome Allows Dissection of Ribosome Pausing
546
Besides uORFs, CDS regions also possess 30-nt phased 5′-truncated mRNA
547
ends (Figure 6). Some regions likely encode nascent peptides which can stall
548
ribosomes during translation elongation as the MTO1 region in CGS1 or are
549
upstream of structured regions which can block the movement of ribosomes
550
(Onouchi et al., 2005). The one with the last peak of 5′-truncated RNA ends
551
located 16 nt upstream of main ORF stop codons as in the degradation pattern
552
observed in CPuORFs may cause ribosome pausing during translation
553
termination. A previous study showed that the binding of EUKARYOTIC
554
RELEASE FACTOR 1 (eRF1) to three types of stop codons causes a
555
conformational change in the ribosome and a 2-nt shift toward the 3′ end of
556
mRNA (Kryuchkova et al., 2013). Because the position 17 nt upstream of the
557
main ORF stop codon is in frame with the 3-nt periodicity observed in the
558
CDSs (Figure 1A), the specific accumulation of 5′-truncated mRNA ends at this
559
position suggests the pausing of a ribosome with the A site at the stop codon.
560
On the other hand, the 5′-truncated mRNA ends peak 16 nt upstream of the
21
561
stop codon may imply the stalling of ribosomes after the conformational
562
change of ribosomes induced by the binding of eRF1. The integration of the
563
RNA degradome data, protein sequences and RNA structures will bring new
564
insights into the regulation of ribosome pausing.
565
566
Exploration of Plant miRNA-mediated Translational Repression with RNA
567
Degradome
568
Unlike animal miRNAs, which mainly target the 3′ UTR, the majority of plant
569
miRNAs bind to their target CDSs through nearly perfect base pairing.
570
Although cleavage sites in the middle of plant miRNA target sites have been
571
extensively validated by degradome data in many species, translational
572
repression mediated by plant miRNAs has also been demonstrated through
573
multiple approaches. Previous analysis of Arabidopsis Ribo-Seq data showed
574
that miRNA targets have lower translational efficiency compared to non-miRNA
575
targets (Liu et al., 2013). However, no preferential accumulation of ribosome
576
footprints was observed in the region upstream of miRNA target sites in
577
Arabidopsis Ribo-Seq data (Liu et al., 2013). Because Ribo-Seq has some
578
drawbacks in detecting stacked ribosomes or ribosomes pausing at the
579
translocation stage as we have discussed, we used RNA degradome data to
580
reexamine whether plant miRNAs can directly stall ribosomes . Consistent with
581
the previous observation in Ribo-Seq data (Liu et al., 2013), no site-specific
582
enrichment of 5′-truncated RNA ends was found in the region upstream of
583
Arabidopsis and rice miRNA target sites (Figure 7). This strengthens the notion
584
that directly blocking ribosome movement through the binding of RISC
585
contributes little to plant miRNA-mediated translational repression in planta.
586
Nevertheless, the binding of AGO7 to a well characterized non-cleavable
22
587
target site of miR390 seems to hinder the movement of ribosomes and cause
588
ribosome stacking as the upstream regions show signatures of stacked
589
ribosomes in the same way as bZIP CPuORFs (Figures 2 and 8). The same
590
degradation pattern corresponding to ribosome stacking among the three
591
Arabidopsis TAS3 genes suggests that this unique configuration of ORFs and
592
non-cleavable miR390 target sites might be crucial for tasiRNA production.
593
Although the function of ORFs upstream of the miR390 non-cleavable site has
594
not been tested, positioning a target site of miR173, another well-known
595
tasiRNA trigger bound to AGO1 (Cuperus et al., 2010), within 10 nt of a stop
596
codon of an upstream ORF was shown to enhance the production of artificial
597
tasiRNA (Zhang et al., 2012). Moreover, a recently published paper also
598
demonstrated that an ORF surrounding the miR173 target site on TAS2 is
599
translated and plays a crucial role in tasiRNA production (Yoshikawa et al.,
600
2016). That signatures of stacked ribosomes exist close to the miR390
601
non-cleavable target site revealed in this study strongly suggests that
602
translation contributes to tasiRNA biogenesis through a conserved mechanism
603
regardless of miRNA triggers or AGO involved.
604
A bottleneck in the development of RNA degradome data applications is the
605
complex composition of mRNA degradation intermediates, complicating
606
interpretation. The discovery of ribosome footprints in RNA degradome data
607
opens up the possibility of new applications of such data in post-transcriptional
608
gene regulations beyond the validation of miRNA-guided cleavage. It should
609
be possible to apply the RNA degradome data analyses demonstrated in this
610
study to many other plant species that have RNA degradome data available,
611
enabling deeper insights into ribosome stalling and mechanisms of RNA
612
decay.
23
613
614
METHODS
615
Plant Materials and Growth Conditions
616
Arabidopsis thaliana (ecotype Col-0) used in this study was grown in soil or on
617
0.8% Bacto-agar plates containing half-strength Murashige and Skoog (MS)
618
medium (pH 5.7) and 1% sucrose under a 16/8-h light/dark cycle with an
619
irradiance of 50-90 μmol photons m–2 s–1 at 22°C. For the generation of PARE
620
data, 11-d-old seedlings and inflorescences of wild-type A. thaliana were used
621
for total RNA isolation. For the analysis of 5′-truncated RNA ends generated
622
from bZIP2 and bZIP11 by the modified RLM 5′ RACE assay, 10-d-old
623
wild-type seedlings were transferred into liquid half-strength MS medium with
624
or without 6% sucrose. After incubation in growth chambers with rotary
625
shaking at 40 rpm under constant light for 24 h, the seedlings were collected
626
for RNA extraction. The inflorescences of Arabidopsis wild type, ago7
627
(GK-824A08-025510), and rdr6 (CS24285) were harvested for the analysis of
628
5′-truncated RNA ends generated from TAS3a with a modified RLM 5′ RACE
629
assay and for PARE library construction.
630
631
PARE Library Construction and Sequencing
632
Total RNA isolated by PureLink Plant RNA Reagent (Thermo Fisher) and
633
MaxTract high-density gel tubes (Qiagen) was used for PARE library
634
construction following the protocol published previously (Zhai et al., 2014).
635
PARE libraries were constructed with about 80 μg total RNA and then
636
sequenced on the Illumina HiSeq 2500 platform.
637
638
Modified RLM 5′ RACE Assay
24
639
Modified RLM 5′ RACE assay was performed to detect 5′-truncated RNA ends
640
using GeneRacer Kit (Thermo Fisher). First, 2-3 μg of total RNA isolated by
641
PureLink Plant RNA Reagent and MaxTract high-density gel tubes was ligated
642
with the 5′ RNA adapter and then reversely transcribed with the oligo-dT primer.
643
Next, cDNA was used as the template for PCR analysis with a GeneRacer 5′
644
primer and a gene-specific primer. Nested PCR was performed with a
645
GeneRacer 5′ nested primer and a gene-specific nested primer if no PCR
646
products were detected in the primary PCR. Amplified products of expected
647
size were gel purified, cloned into pJET1.2/blunt Cloning Vector or
648
pCR4-TOPO TA Vector (Thermo Fisher) and sequenced. A target of miR159,
649
MYB65, was included as the positive control for the modified RLM 5′ RACE
650
assay. The primers are listed in Supplemental Table 1.
651
652
Analysis of 5′-truncated RNA End Distribution
653
In addition to the in-house PARE data of Arabidopsis, previously published
654
PARE data of Arabidopsis, rice, and soybean downloaded from the Gene
655
Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) were also
656
analyzed in this study. Accession numbers are given at the end of Methods.
657
Trimmed reads were mapped to the corresponding genomes or gene
658
sequences downloaded from TAIR (https://www.arabidopsis.org/; TAIR 10),
659
the MSU Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/;
660
Release 6.1), and Phytozome (https://phytozome.jgi.doe.gov/; Phytozome
661
v11.0
662
(http://bowtie-bio.sourceforge.net/; v1.0.0). Only perfectly matched 20-nt
663
sequences were used in the following metagene analyses and sequences of
664
low complexity (repeats ≥ 15 nt) or with ≥ 10 hits in the genome were excluded.
:
Gmax_275_Wm82.a2.v1
25
)
with
Bowtie
665
The abundance of PARE sequences was assigned to the position
666
corresponding to the first nucleotide of the sequence. Known Arabidopsis and
667
rice CPuORF and miRNA target sites were retrieved from previous reports
668
(Hayden and Jorgensen, 2007; Zheng et al., 2012).
669
For metagene analysis of PARE reads in the regions flanking the start
670
codon and the stop codon of annotated CDSs, the abundance of PARE reads
671
at each position in the defined regions on a transcript was first normalized by
672
dividing the value by the sum of PARE reads starting in the defined region.
673
Then the relative abundance at each position across the defined region was
674
calculated as the sum of normalized abundance of PARE reads starting at the
675
same position for all genes. For heat maps of PARE read distribution in regions
676
upstream of uORF stop codons or miRNA guided cleavage sites, we
677
normalized the abundance of PARE reads at each position by dividing the
678
value by the sum of PARE reads starting in a 31-nt window flanking the
679
indicated position. The distribution of normalized PARE abundance was then
680
clustered using Ward’s method with R package (https://www.r-project.org/;
681
version 2.15.2) and displayed as heat maps.
682
683
Ribo-Seq Data Analysis
684
Previously published Ribo-Seq data sets of Arabidopsis were downloaded
685
from the GEO database. Accession numbers are given at the end of Methods.
686
Trimmed reads of length ≥ 20 nt were mapped to gene sequences downloaded
687
from TAIR with Bowtie. Two-nt mismatches were allowed for mapping
688
Ribo-Seq reads of normoxia seedlings, whereas perfect matches were used in
689
the mapping of Ribo-Seq reads of light-treated seedlings. The abundance of
690
Ribo-Seq sequences was assigned to the position corresponding to the first
26
691
nucleotide of the sequence.
692
693
Identification of uORFs with Footprints of Stacked Ribosomes
694
RNA ends protected by monosome and disome pausing at uORF stop codons
695
should result in PARE peaks at positions 16 and 46 nt upstream of the stop
696
codon. A custom Perl script was used to identify uORFs possessing this
697
degradation signature with in-house Arabidopsis PARE data (Supplemental
698
Script 1). First, the sequences of Arabidopsis 5′ UTRs based on TAIR10
699
annotation were used to predict uORFs by looking for an ATG codon paired
700
with the nearest in-frame stop codon. Next, PARE reads were mapped to the
701
predicted uORFs by this Perl script. Candidates of uORFs with a signature of
702
ribosome stacking at the stop codon were selected based on the following
703
criteria. To capture the signature of ribosome stacking, the distribution of PARE
704
reads was evaluated in two 31-nt regions upstream of the stop codon. The first
705
region spans positions -1 to -31 and the second region spans positions -32 to
706
-62, with the first nucleotide of the uORF stop codon is set to 0. In the first
707
region, the most abundant peak (the first major peak) was required to be at
708
position -16 or -17 and with the number of raw reads ≥3. In the second region,
709
the most abundant peak (the second major peak) was required to be located at
710
position -45, -46 or -47. Moreover, the abundance of the major peaks was
711
required to be at least 2-fold higher than that of the second most abundant
712
peak in the same region but outside the possible positions for major peaks.
713
714
Identification of Degradation Signatures Representing Stacked
715
Ribosomes in the CDSs
716
To identify degradation signatures representing stacked ribosomes in the
27
717
CDSs, we first mapped trimmed PARE sequences to Arabidopsis CDSs based
718
on TAIR10 annotation with Bowtie Because the protected degradation termini
719
caused by three stacked ribosomes are three predominant PARE peaks
720
separated by about 30 nt, we calculated two values, Peak_Abundance and
721
Peak_Index, to evaluate the predominance of PARE reads accumulated at
722
each position. Peak_Abundance was defined as the sum of PARE reads
723
starting at the indicated position together with the positions 1 nt upstream and
724
downstream. Peak_Index was calculated by dividing the Peak_Abundance by
725
the total PARE reads starting in a 31-nt window flanking the indicated position.
726
Positions with Peak_Index ≥ 0.3 were selected as the first major peak and
727
were reset to 0 in further analysis. The second and third major peaks were
728
required to be at the regions between -29 and -31, and -59 and -61 with
729
Peak_Index ≥ 0.3. In addition, the abundance of three major peaks had to be
730
the highest in the region for the calculation of Peak_Index and with raw reads ≥
731
3. The abundance of the three major peaks also needed to be 2-fold higher
732
than that of the second most abundant peak falling in the 31-nt window but
733
outside the possible positions for major peaks. To ensure that the degradation
734
signature caused by stacked ribosomes was prominent among all degradation
735
events within a gene, a region was selected only if all three major peaks were
736
ranked in the top 1% of all positions with regard to PARE abundance. To
737
eliminate the phased regions occurring by chance, the candidates uncovered
738
from only a single sample were removed. The in-house Perl script for PARE
739
peak analysis in CDSs is provided as Supplemental Script 2. The same criteria
740
were applied for the phasing analysis of PARE peaks for intervals from 20 to
741
40 nt.
742
28
743
Generation of Transgenic Lines
744
A 1.2 kb DNA fragment of Arabidopsis CIPK6 promoter region plus 5′ UTR was
745
cloned into gateway vector pHGWFS7 upstream a GUS reporter gene through
746
Gateway LR Clonase II Enzyme mix (Thermo Fisher). Site-directed
747
mutagenesis was applied to change the start codon of CIPK6 CPuORF to a
748
stop codon. The two constructs with WT uORF and ΔuORF were transformed
749
into wild-type Arabidopsis through the floral dip method (Zhang et al., 2006).
750
Primers used to clone the 1.2kb CIPK6 promoter fragment are listed in
751
Supplemental Table 1.
752
753
GUS Activity Assay and GUS Staining
754
The extract of ground tissues extracted by GUS extraction buffer (50 mM
755
NaHPO4 pH 7.0, 10 mM 2-mercaptoethanol, 10 mM Na2EDTA, 0.1% sodium
756
lauryl sarcosine and 0.1% Triton X-100) was mixed with MUG buffer
757
(4-methylumbellifery β-D-glucuronide) at 37°C for 20 min, and then the
758
reaction was terminated by adding stop buffer (0.2 M sodium carbonate). The
759
fluorescence intensity of 4-methylumbelliferone (4MU) was measured by
760
fluorometer at 450 nm when excited at 365 nm. The amount of 4MU was
761
calculated with the standard curve and then normalized to the amount of total
762
protein which was measured using the Bradford dye-binding method with the
763
BioRad Labs protein assay kit. The GUS transgenic plants were stained in
764
GUS staining solution (0.1 M NaPO4 pH 7.0, 10 mM EDTA, 0.1% Triton X-100,
765
1 mM K3Fe(CN)6 and 2 mM X-Gluc) at 37°C overnight. After staining, samples
766
were washed with 50% ethanol until chlorophyll was removed, which took
767
either overnight or several days depending on the tissue. Then the stained
768
samples were observed under a stereomicroscope (SteREO Lumar.V12,
29
769
ZESIS) and photographed with a digital camera (AxioCam MRc, ZESIS).
770
771
Quantitative RT-PCR (qRT-PCR)
772
2 µg of total RNA of was used as a template for reverse transcription with
773
ToolsQuant II Fast RT kit (BIOTOOLS). The resulting cDNA was diluted 20-fold
774
and 5 µL was used for qRT-PCR in a 20 µL reaction with SYBR Green PCR
775
Master Mix (Applied Biosystems) on a 7500 Fast Real-Time PCR System
776
(Applied Biosystems) using the following program: 20 s at 95°C, followed by 40
777
cycles of 3 s at 95°C and 30 s at 60°C, with an additional melt curve stage
778
consisting of 15 s at 95°C, 1 min at 60°C, and 15 s at 95°C. TAS3a and GUS
779
expression levels were normalized to the level of UBQ5 expression and were
780
averaged from at least three independent biological samples, followed by
781
normalizing to the corresponding value of wild type. The primers used are
782
listed in Supplemental Table 1.
783
784
Protoplast Transient Assay
785
For construction of reporter plasmids, MYB34 and MYB51 5′ UTRs were
786
amplified from Arabidopsis genomic DNA and cloned into pJD301 between the
787
cauliflower mosaic virus 35S promoter and the firefly luciferase (LUC) coding
788
region using the NcoI restriction site. To abolish MYB34 and MYB51 uORFs,
789
the start codon of uORFs was converted into a stop codon by site-directed
790
mutagenesis. Primers used in the cloning of MYB34 and MYB51 5′ UTR
791
sequences are listed in Supplemental Table 1. Arabidopsis mesophyll
792
protoplasts were isolated from 3 to 4-week-old rosette leaves following the
793
method described previously (Wu et al., 2009). Equal amounts (20 µg) of
794
reporter plasmids and internal control plasmids containing the 35S promoter
30
795
driving a GUS gene were cotransfected into 105 protoplasts in a PEG solution
796
(40% polyethylene glycol 4000, 0.2 M mannitol and 0.1 M CaCl2) at room
797
temperature for 5-10 min. The transfected protoplasts were incubated at 22°C
798
in the dark for 16 h and then lysed with Cell Culture Lysis Reagent (CCLR,
799
Promega). LUC activity was measured with a Luciferase Assay System
800
(Promega) according to the manufacturer’s instructions and normalized to
801
GUS activity which was measured as described above.
802
803
RNA Gel Blot of Small RNA
804
Small RNA blot analysis was performed as described previously (Lee et al.,
805
2015). The probes used in the detection of small RNAs derived from TAS3 and
806
U6 are listed in Supplemental Table 1.
807
808
Alignment of Peptides Encoded by MYB34 and MYB51 uORFs
809
The sequences of MYB34 and MYB51 genes in different plant species were
810
identified by a BLASTP search and downloaded from the Phytozome database
811
(https://phytozome.jgi.doe.gov). The 5′ UTR sequences of orthologous genes
812
retrieved for uORF analysis with Serial Cloner (http://serialbasics.free.fr/;
813
version 2.6) are listed in Supplemental Data Set 1. Peptides encoded by
814
MYB34 and MYB51 uORF were then aligned with the use of Vector NTI
815
software (Thermo Fisher).
816
817
Accession Numbers
818
The PARE data generated in this study as well as public PARE data are
819
available in the GEO database under series GSE77549 (Arabidopsis in-house
820
PARE data), and accession numbers GSM280226 (public Arabidopsis
31
821
inflorescence PARE data), GSM647200 (public soybean seed PARE data),
822
GSM434596 (public rice seedling PARE data) and GSM476257 (public rice
823
young inflorescence PARE data). The Arabidopsis Ribo-Seq data sets used in
824
this study are available in the GEO database under the accession numbers
825
GSM1226369
826
seedlings). Sequences of individual genes used in PARE data analysis or
827
functional assays and mutants used can be found in the TAIR or Phytozome
828
databases under
829
Ath-SAMDC, AT2G18160 for Ath-bZIP2, AT4G34590 for Ath-bZIP11,
830
AT3G62420 for Ath-bZIP53, AT4G30960 for Ath-CIPK6, Glyma09g14090 for
831
Gly-CIPK6, AT5G60890 for Ath-MYB34, AT1G18570 for Ath-MYB51,
832
AT3G01120 for Ath-CGS1, AT3G17185 for Ath-TAS3a, AT5G49615 for
833
Ath-TAS3b, AT5G57735 for Ath-TAS3c, AT1G69440 for Ath-ago7, and
834
AT3G49500 for Ath-rdr6.
(light-treated
the
seedlings)
following
and
accession
GSM1224475
(normoxia
numbers: AT3G02470
for
835
836
SUPPLEMENTAL DATA
837
Supplemental Figure 1. 5′-Truncated RNA Ends Show a 3-nt Periodicity and
838
Significant Frame Bias in the CDS.
839
Supplemental Figure 2. Overaccumulation of 5′-Truncated RNA Ends at the 3′
840
End of Arabidopsis SAMDC uORF.
841
Supplemental Figure 3. The Comparison of PARE and Ribo-Seq Read
842
Distribution in Arabidopsis bZIP uORFs.
843
Supplemental Figure 4. Site-specific Enrichment of 5'-Truncated RNA Ends
844
in Arabidopsis CPuORFs.
845
Supplemental Figure 5. PARE Read Distribution in Arabidopsis uORFs with
846
RNA Degradation Signatures Representing Ribosome Stacking at Stop
32
847
Codons.
848
Supplemental Figure 6. The Comparison of PARE and Ribo-Seq Read
849
Distribution in Arabidopsis CIPK6, MYB34 and MYB51 uORFs.
850
Supplemental Figure 7. Negative Regulation of CIPK6 uORF in Reporter
851
Gene Expression in Various Tissues.
852
Supplemental Figure 8. The Comparison of PARE Read Distribution with the
853
5′ Ends of the Degradation Intermediates Previously Identified around the
854
MTO1 Region of CGS1.
855
Supplemental Figure 9. Phasing Analysis of PARE Peaks in the CDS.
856
Supplemental Figure 10. The Comparison of PARE and Ribo-Seq Read
857
Distribution in CDS Regions with 30-nt Phased PARE Peaks.
858
Supplemental Figure 11. The Comparison of PARE and Ribo-Seq Read
859
Distribution in Arabidopsis TAS3 Genes.
860
Supplemental Figure 12. Three Arabidopsis TAS3 Genes Accumulate
861
AGO7-dependent but RDR6-indepdennt Phased 5′-Truncated RNA Ends
862
Upstream of Non-Cleavable miR390 Target Sites.
863
Supplemental Table 1. Sequences of Primers for Cloning, Modified RLM 5′
864
RACE, qRT-PCR and Probes for RNA Gel Blot Analysis.
865
Supplemental Data Set 1. Sequences for the Analysis of Conserved uORFs
866
in MYB34 and MYB51.
867
Supplemental Script 1. In-house Perl script for PARE peak analysis in uORF.
868
Supplemental Script 2. In-house Perl script for PARE peak analysis in CDSs.
869
870
ACKNOWLEDGEMENTS
871
We thank Shu-Hsing Wu and Tzyy-Jen Chiou of Academia Sinica for helpful
872
discussions and Ming-Che Shih for supporting Hsiao-Chun Chou. We also
33
873
thank the Academia Sinica Agricultural and Biotechnology Research Center
874
core facilities for help with transgenic plants and protoplast assays and
875
Miranda Loney for English editing of this paper. This work was supported by
876
Academia Sinica.
877
878
AUTHOR CONTRIBUTIONS
879
H.-M.C. designed the research. C.-Y.H., H.-C.C. and H.-M.C. performed the
880
sequence data analyses. W.-C.L., H.-C.C., A.-P.C. and S.-J.C. carried out
881
experiments. C.-Y.H., W.-C.L. and H.-M.C. wrote the paper. All authors read
882
and approved the final manuscript.
883
884
885
886
FIGURE LEGENDS
Figure 1. 5′-Truncated RNA Ends Show a 3-nt Periodicity and Frame Bias
in the CDS.
887
888
889
(A) The positional distribution of 5′ ends of truncated RNA mapped to the
regions surrounding the start codon and the stop codon of Arabidopsis, rice
and soybean CDS. PARE data of Arabidopsis seedlings and inflorescences
890
891
892
893
were generated by this study and PARE data of rice inflorescences and
soybean seeds were published by Zhou et al. (2010) and Song et al. (2011).
Blue bars indicate positions falling in the translational frame (frame 1) of
annotated CDS if 5′-truncated ends represent the 5′ edge of a ribosome and
894
895
896
897
the distance from the 5′ edge of a ribosome to the first base of the A site is 17
nt. Red arrowheads beneath the graphs represent the first nt in the start codon
(left side) or the stop codon (right side). The illustration at the bottom shows
the size of an mRNA fragment protected by a plant ribosome and the position
898
899
900
of ribosomes decoding start and stop codon. CDS, coding sequence (dark
blue); UTR, untranslated region (light blue); E, the exit site; P, the peptidyl site;
A, the aminoacyl site.
901
902
903
904
(B) The proportion of 5′-truncated RNA ends mapped to complete CDS in all
three frames. frame 1, the translational frame of TAIR annotated CDS; frames
2 and 3, the frames offset +1 and +2 from frame 1.
905
Figure 2. bZIP uORFs Accumulate a Ladder of 5′-Truncated RNA Ends at
34
906
907
908
30-nt Intervals.
(A) The positional distribution of 5′ ends of truncated RNA generated by PARE
and ribosome-protected mRNA fragments generated by Ribo-Seq in
909
910
911
912
Arabidopsis bZIP2, bZIP11, and bZIP53 5′ UTRs. The PARE data of
Arabidopsis inflorescences and the Ribo-Seq data of light-treated seedlings
were retrieved from the data sets published by German et al. (2008) and Liu et
al. (2013) respectively. The PARE data of Arabidopsis seedlings were
913
914
915
916
generated by this study. Regions of CPuORFs are shown as dark blue lines
under the graphs. The PARE peaks at positions 16, 46, and 76 nt upstream of
the uORF stop codon are highlighted in red. TP10M, tags per 10 million;
TP50M, tags per 50 million.
917
918
919
920
(B) Modified RLM 5′ RACE assays of bZIP2 and bZIP11 transcripts in
Arabidopsis seedlings treated with or without 6% sucrose. MYB65, a target of
miR159, was used as a positive control for the modified RLM 5′ RACE assay.
The brackets indicate the PCR products excised and cloned for Sanger
921
922
923
924
sequencing (left panels). In total, 36 and 16 clones were sequenced for bZIP2
and bZIP11, respectively. The positional distribution of 5′-truncated RNA ends
increased under 6% sucrose treatments is shown for the bZIP2 and bZIP11 5′
UTRs (right panels). Regions of CPuORFs are shown as dark blue lines under
925
the graphs. TSS: transcriptional start site.
926
927
928
929
930
931
932
Figure 3. Site-specific Enrichment of 5′-Truncated RNA Ends is Evident in
CPuORFs.
(A) Clustered heat maps of 5′-truncated RNA ends mapped in a 55-nt region
upstream of the stop codon of CPuORFs and predicted uORFs in wild-type
Arabidopsis inflorescences and rice seedlings. Predicted uORFs overlapping
CPuORFs are not included in the heat maps of predicted uORFs. Arabidopsis
933
934
and rice PARE data used in this analysis were published by German et al.
(2008) and Li et al. (2010) respectively.
935
936
(B) Clustered heat maps of 5′ RNA ends protected by ribosomes in a 55-nt
region upstream of the stop codon of CPuORFs and predicted uORFs in
937
938
939
wild-type Arabidopsis. The Ribo-Seq data sets of light-treated and normoxic (in
air) seedlings used in this analysis were published by Liu et al. (2013), and
Juntawong et al. (2014), respectively.
940
941
942
943
In (A) and (B), the first nt of the stop codon is assigned position 0, and the
color of data points represents the Peak_Index value, which is calculated by
dividing the number of PARE or Ribo-Seq reads starting at the position
indicated by the number of total reads in a 31-nt flanking region. The numbers
35
944
945
946
of annotated CPuORFs and predicted uORFs (indicated in parentheses) and
uORFs included in heat maps are shown above the heat maps.
947
Figure 4. CIPK6 CPuORF Possesses Footprints of Stacked Ribosomes
948
949
950
and Represses Downstream ORF Expression in Various Tissues.
(A) The positional distribution of 5′ ends of truncated RNA generated by PARE
and ribosome-protected mRNA fragments generated by Ribo-Seq in
951
952
953
954
Arabidopsis CIPK6 5′ UTR. The PARE data of Arabidopsis inflorescences and
the Ribo-Seq data of light-treated seedlings plotted were retrieved from the
data sets published by German et al. (2008), and Liu et al. (2013), respectively.
TP10M, tags per 10 million.
955
956
(B) The positional distribution of 5′-truncated RNA ends generated by PARE in
soybean CIPK6 5′ UTR. The soybean PARE data plotted were retrieved from
957
958
the data set published by Song et al. (2011). In (A) and (B), regions of
CPuORFs are shown as dark blue lines under the graphs and the first nt of the
959
960
stop codon is assigned position 0. The PARE or Ribo-Seq peaks at positions
16, 46, and 76 nt upstream of the uORF stop codon are highlighted in red.
961
962
(C) Histochemical staining of randomly selected Arabidopsis T1 transgenic
plants carrying a GUS reporter gene driven by Arabidopsis CIPK6 promoter
963
964
965
with
wild-type
(CIPK6pro:UTR(WT):GUS)
and
deleted
uORF
(CIPK6pro:UTR(ΔuORF):GUS). The red arrowhead indicates the site mutated.
Bar = 1 cm.
966
967
(D) Comparison of GUS activity between WT and ΔuORF transgenic plants.
The amount of total protein was used for the normalization of GUS activity.
968
969
(E) Comparison of GUS mRNA level between WT and ΔuORF transgenic
plants by qRT-PCR. The mRNA level of UBQ5 was used for the normalization
970
971
972
973
974
of GUS mRNA level. In (D) and (E), each bar represents the mean of
measurements derived from five independent T1 transgenic plants ± SE
relative to the measurement of WT. *P < 0.05; ns, no significant difference
(two-tailed student’s t-test, n = 5).
975
Figure 5. Lineage-specific CPuORFs in MYB34 and MYB51 Generate
976
977
978
979
980
981
5′-Truncated RNA Ends Separated in 30 nt and Negatively Regulate
Downstream ORF Expression.
(A) The positional distribution of 5′ ends of truncated RNA generated by PARE
and ribosome-protected mRNA fragments generated by Ribo-Seq in the 5′
UTR of MYB34 and MYB51. The PARE data of seedlings were generated by
this study and the Ribo-Seq data plotted were retrieved from the data set
36
982
983
984
published by Liu et al. (2013). Regions of CPuORFs are shown as dark blue
lines under the graphs and the first nt of the stop codon is assigned position 0.
The PARE or Ribo-Seq peaks at positions 16 and 46 upstream of the uORF
985
stop codon are highlighted in red. TP50M, tags per 50 million.
986
987
988
(B) Alignment of peptides encoded by MYB34 and MYB51 uORFs in the
Brassicaceae family. Ath, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Bra,
Brassica rapa; Bst, Boechera stricta; Cru, Capsella rubella; Esa, Eutrema
989
990
salsugineum. The alignment is colored according to residue conservation: red,
identical residues; orange, conserved residues; pink, block of similar residues.
991
992
(C) Transient expression assays of MYB34 and MYB51 uORF regulatory
function using LUC reporter constructs in protoplasts. The reporter constructs
993
994
995
996
of wild-type (WT) and deleted uORF (ΔuORF) are illustrated. The red
arrowhead indicates the site mutated. The LUC reporter constructs were
co-transfected with a control of a GUS gene driven by a 35S promoter. The
LUC activity was first normalized to GUS activity and then to the value of WT
997
998
999
construct. Each bar represents the mean of measurements derived from six
independent protoplast transfections ± SE relative to the measurement of WT.
*P < 0.05; ns, no significant difference (two-tailed student’s t-test, n = 6).
1000
1001
Figure 6. Analysis of PARE Data Reveals CDS Regions Potentially
1002
1003
1004
Associated with Stacked Ribosomes.
The positional distribution of 5′ ends of truncated RNA generated by PARE and
ribosome-protected mRNA fragments generated by Ribo-Seq in CDS regions
1005
1006
1007
1008
potentially associated with stacked ribosomes. PARE data of Arabidopsis
inflorescences plotted were generated by this study while the Ribo-Seq data
plotted were retrieved from the data set published by Liu et al. (2013). The
30-nt phased PARE peaks and the corresponding Ribo-Seq peaks are
1009
1010
highlighted in red with coordinates indicated above. TP50M tags per 50 million.
1011
1012
1013
1014
1015
1016
Figure 7. No Site-specific Enrichment of 5′-Truncated RNA Ends is
Detected in the Proximal Region Upstream of miRNA-guided Cleavage
Sites.
Clustered heat maps of 5′-truncated RNA ends mapped to a 55-nt region
upstream of miRNA-guided cleavage sites in seedlings and inflorescences of
Arabidopsis and rice. The Arabidopsis PARE data plotted were generated by
1017
1018
1019
this study while the rice PARE data plotted were retrieved from the data sets
published by Li et al. (2010) and Zhou et al. (2010). The presumed
miRNA-guided cleavage site is assigned position 0 and the color represents
37
1020
1021
1022
the value of Peak_Index which is calculated by dividing the reads starting at
the position indicated by the total reads in a 31-nt flanking region. The
numbers of known miRNA target sites (indicated in parentheses) and target
1023
1024
1025
sites possessing PARE reads and included in heat maps are shown above
heat maps.
1026
1027
Figure 8. Arabidopsis TAS3 Genes Accumulate AGO7-dependent but
RDR6-independent Phased 5′-Truncated RNA Ends Upstream of
1028
1029
1030
Non-cleavable miR390 target sites.
(A) The distribution of 5′ ends of truncated RNA generated by PARE and
ribosome-protected mRNA fragments generated by Ribo-Seq in three
1031
1032
1033
1034
Arabidopsis TAS3 genes. The PARE data of seedlings plotted were generated
by this study and the Ribo-Seq data of light-treated seedlings plotted were
retrieved from the data set published by Liu et al. (2013). The predominant
PARE and Ribo-Seq peaks are highlighted in red and marked with their
1035
1036
distances to the first nt of non-cleavable miR390 target sites. TP50M, tags per
50 million.
1037
1038
(B) Comparison of 5′-truncated RNA ends generated in the region upstream of
the non-cleavable miR390 target site of TAS3a in wild type (WT), rdr6 and
1039
1040
1041
1042
ago7 by the modified RLM 5′ RACE assay. The bracket indicates the PCR
products excised and cloned for Sanger sequencing (left panel). In total, 12
and 13 clones were sequenced for WT and rdr6 respectively. The positional
distribution of 5′-truncated RNA ends revealed by the modified RLM 5′ RACE
1043
1044
assay is plotted relative to the non-cleavable miR390 target site (right panel).
The cleavage target of miR159, MYB65, is used as a positive control for the
1045
1046
modified RLM 5′ RACE assay. In (A) and (B), the first nt of the non-cleavable
miR390 target site is assigned position 0.
1047
1048
1049
1050
(C) Comparison of TAS3a transcript levels in WT, rdr6 and ago7 by qRT-PCR.
The mRNA level of UBQ5 was used for the normalization of TAS3a mRNA
levels. Each bar represents the mean of measurements derived from four
biological replicates ± SE relative to the measurement of WT. *P < 0.05; **P <
1051
0.01 (two-tailed student’s t-test, n = 4).
1052
1053
1054
(D) Comparison of TAS3a tasiRNA produced in WT, rdr6 and ago7 by RNA gel
blot with U6 as the loading control. Numbers to the left of the blot show sizes in
nt. Antisense DNA oligonucleotides were used as probes for U6 and a TAS3
1055
1056
tasiRNA.
1057
Figure 9. Schematic Representation of RNA Degradation Fragments
38
1058
1059
1060
Protected by Stacked Ribosomes in Distinct Regions.
(A) Degradation signatures caused by stacked ribosomes upstream of uORF
stop codons. Position 0 is the first nt of the uORF stop codon.
1061
1062
(B) Degradation signatures caused by stacked ribosomes in the CDS. Position
0 is the 3′ edge of the most 3′ stalled ribosome.
1063
1064
(C) Degradation signatures caused by stacked ribosomes upstream of a
non-cleavable miRNA target site bound by AGO7. Position 0 is the first nt of
1065
1066
1067
1068
the miRNA target site.
Predominant peaks of 5′-truncated RNA ends are indicated with vertical lines
and their distances to the putative sites causing ribosome stalling (highlighted
in red) or to the 3′ edge of the most 3′ stalled ribosome (highlighted in blue) are
1069
1070
1071
marked below. The height of vertical lines represents the abundance of
5′-truncated RNA ends. 60S, 60S subunit of ribosome; 40S, 40S subunit of
ribosome.
1072
1073
Table 1. Arabidopsis uORFs with Two Predominant PARE Peaks
1074
Representing Two tandem Ribosomes Stacking at Stop Codons
Gene Model
Name
Description
Coordinates of
Coordinates of
uORF
PARE Peak
PARE Library
CPuORFs in TAIR10
AT1G75390.1
bZIP44
Basic leucine-zipper 44
258-380
336, 365
INF
AT2G18160.1
bZIP2
Basic leucine-zipper 2
201-323
279, 308
SD
AT5G01710.1
Methyltransferase
142-252
207, 237
INF
bZIP1
Basic leucine-zipper 1
229-303
258, 288
INF
AT1G18570.1
MYB51
MYB domain protein 51
156-221
176, 206
SD, INF
AT2G01930.2
BPC1
Basic pentacysteine1
5-310
265, 295
SD
AT4G30960.1
CIPK6
CBL-interacting protein kinase
132-227
182, 212
SD, INF
AT5G60890.1
MYB34
MYB domain protein 34
52-150
106, 135
SD
AT5G49450.1
Novel uORFs
1075
1076
SD: seedlings; INF: inflorescences
1077
39
1078
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1079
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Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants
Cheng−Yu Hou, Wen−Chi Lee, Hsiao−Chun Chou, Ai−Ping Chen, Shu-Jen Chou and Ho-Ming Chen
Plant Cell; originally published online October 14, 2016;
DOI 10.1105/tpc.16.00295
This information is current as of June 17, 2017
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