Plant Cell Advance Publication. Published on October 14, 2016, doi:10.1105/tpc.16.00295
LARGE-SCALE BIOLOGY ARTICLE
Global Analysis of Truncated RNA Ends Reveals New Insights into
Ribosome Stalling in Plants
Cheng‐Yu Houa, Wen‐Chi Leea, Hsiao‐Chun Choua, b, Ai‐Ping Chenc, Shu‐Jen Chouc
and Ho‐Ming Chena1
aAgricultural
Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan
bInstitute
of Plant Biology, National Taiwan University, Taipei, 10617, Taiwan
cInstitute
of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan
d
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
(www.plantcell.org) is: Ho-Ming Chen ([email protected]).
One-sentence summary: In a global analysis of free 5' mRNA ends, this study shows that
the plant RNA degradome contains in vivo ribosome footprints and is useful to study
ribosome stalling during translation.
ABSTRACT
High-throughput approaches for profiling the 5′ ends of RNA degradation intermediates on a
genome-wide scale are frequently applied to analyze and validate cleavage sites guided by
microRNAs (miRNAs). However, the complexity of the RNA 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 (Arabidopsis thaliana), rice (Oryza sativa) and soybean
(Glycine max). Based on this 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 frame. By examining conserved peptide upstream open reading frames
(uORFs) of Arabidopsis and rice, we found a predominance of 5′ termini of RNA degradation
intermediates that were separated by a length equal to a ribosome-protected mRNA
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 Arabidopsis ARGONAUTE7
to a non-cleavable target site of miR390 might directly hinder ribosome movement. This
work demonstrates an alternative use of RNA degradome data in the study of ribosome
stalling.
©2016 American Society of Plant Biologists. All Rights Reserved
1
INTRODUCTION
2
Steady-state levels of RNA are controlled by relative rates of transcription and RNA
3
degradation. Most mRNAs in eukaryotes possess a 7-methylguanosine cap at the 5′
4
terminus and a poly(A) tail at the 3′ terminus which are crucial for translation and RNA
5
stability. The loss of the 5′ cap or the 3′ poly(A) tail abolishes mRNA translation and
6
promotes mRNA degradation (Gallie, 1991). Uncapped 5′ ends of mRNAs are
7
degraded by 5′-3′ exoribonucleases (XRNs) whereas deadenylated mRNAs are
8
degraded by the exosome from the 3′ end (Lebreton and Seraphin, 2008; Houseley
9
and Tollervey, 2009). Alternatively, deadenylation can also trigger decapping of mRNA,
10
followed by degradation from the 5′ end (Muhlrad et al., 1994).
11
Translation plays a crucial role in controlling mRNA stability and is required to
12
eliminate aberrant mRNAs through several distinct mechanisms (Shoemaker and
13
Green, 2012). Nonsense-mediated mRNA decay (NMD) is often initiated when a
14
ribosome encounters a premature termination codon (PTC) upstream of an exon
15
junction complex (EJC). The interaction between NMD factors on termination factors
16
and EJCs promotes the degradation of PTC-containing transcripts. The movement of
17
ribosomes on mRNA molecules can be stopped externally by a stable RNA structure
18
or internally by the particular peptide that is encoded by the mRNA. The transcripts
19
associated with stalled ribosomes are degraded by a specialized RNA surveillance
20
pathway called no-go decay, which may result in endonucleolytic cleavage upstream
21
of the stalled ribosomes (Doma and Parker, 2006). Non-stop decay also targets
22
transcripts with stalled ribosomes, but the stalling is due to the lack of an in-frame stop
23
codon (Frischmeyer et al., 2002; van Hoof et al., 2002). Many eukaryotic mRNAs
24
possess short open reading frames (ORFs) in the 5′ untranslated region (UTR) which
25
can be translated and potentially regulate mRNA stability and translation. The stop
26
codon of a translated upstream open reading frame (uORF) might be recognized as a
2
27
PTC and thus initiate NMD. Some uORFs encoding peptides conserved across
28
species have been shown to stall ribosomes at uORF stop codons, resulting in the
29
repression of downstream main ORF translation and acceleration of RNA degradation
30
in a few cases (Gaba et al., 2005; Uchiyama-Kadokura et al., 2014).
31
In eukaryotes, small RNAs of 20 to 30 nt play a key role in regulating gene
32
expression through the RNA interference pathway. Most animal microRNAs (miRNAs)
33
have a seed region, spanning the second to the seventh or the eighth nucleotide,
34
which can base pair perfectly with the 3′ UTR of a target mRNA (Bartel, 2009). The
35
targeting of animal miRNAs is often associated with translation repression,
36
deadenylation and mRNA decay using exoribonucleases. By contrast, plant miRNAs
37
are highly complementary to their targets, and cleavage in the middle of target sites is
38
the major mode of plant miRNA action (Rhoades et al., 2002). However, growing
39
evidence indicates that plant miRNAs can also inhibit the translation of their targets
40
(Brodersen et al., 2008; Iwakawa and Tomari, 2013; Li et al., 2013b; Li et al., 2013c;
41
Liu et al., 2013). In vitro assays of RNA-induced silencing complexes (RISCs)
42
containing Arabidopsis (Arabidopsis thaliana) ARGONAUTE1 (AGO1) demonstrated
43
that plant miRNAs can repress translation initiation uncoupled with deadenylation or
44
mRNA decay (Iwakawa and Tomari, 2013). Furthermore, miRNA binding sites in ORFs
45
can hinder the movement of ribosomes (Iwakawa and Tomari, 2013). In Arabidopsis,
46
miRNA-mediated translation repression occurs in the endoplasmic reticulum and
47
requires the endoplasmic reticulum protein ALTERED MERISTEM PROGRAM1 (Li et
48
al., 2013c).
49
High-throughput approaches for genome-wide profiling of RNA degradation
50
intermediates that possess a free monophosphate at the 5′ terminus have been
51
developed by several groups and are variously named parallel analysis of RNA ends
52
(PARE) (German et al., 2008), degradome sequencing (Addo-Quaye et al., 2008),
3
53
genome-wide mapping of uncapped transcripts (GMUCT) (Gregory et al., 2008), and
54
5′ P sequencing (5Pseq) (Pelechano et al., 2015). Because intact mRNAs generally
55
possess a 5′ cap that blocks their ligation to RNA adaptors, truncated 5′ RNA ends
56
with a free monophosphate can be selectively sequenced by directly ligating poly(A)
57
RNA with RNA adaptors. PARE and degradome sequencing have been widely applied
58
in the identification of small RNA-guided cleavage sites in various plant species (Addo-
59
Quaye et al., 2008; German et al., 2008; Zhou et al., 2010; Shamimuzzaman and
60
Vodkin, 2012; Zhao et al., 2012; Li et al., 2013a). Specific PARE has been developed
61
for the study of plant miRNA processing by specifically amplifying miRNA processing
62
intermediates (Bologna et al., 2013). Although these approaches have been used to
63
profile mRNA degradation intermediates in mutants impaired in XRNs or proteins with
64
endonucleolytic activity (German et al., 2008; Harigaya and Parker, 2012; Schmidt et
65
al., 2015), the interpretations of some results remain challenging because the
66
complexity of the RNA degradome is currently still largely uncharacterized.
67
Previously, we showed that plant RNA degradome data potentially contain footprints
68
of RNA binding proteins in the 3′ UTR (Hou et al., 2014). Pelechano et al. (2015) also
69
demonstrated that yeast 5Pseq data contain in vivo ribosome footprints, the products
70
of co-translation mRNA decay. The change in these ribosome-protected termini
71
captured by 5Pseq could reflect ribosome dynamics without the problems caused by
72
translational inhibitors sometimes used in the generation of in vitro ribosome footprints.
73
Codons associated with paused ribosomes in response to oxidative stress were
74
identified from the analysis of yeast 5Pseq data. Co-translation mRNA decay was also
75
demonstrated to be mediated by XRN4 and involved in the reprogramming of gene
76
expression under heat stress in Arabidopsis (Merret et al., 2013; Merret et al., 2015).
77
Here, we further demonstrate that footprints of ribosomes are widespread in the RNA
78
degradome of various plant species. Our genome-wide analysis of RNA degradation
4
79
fragments revealed uORFs, CDS regions and non-cleavable miRNA target sites that
80
are potentially associated with stacked ribosomes. Our findings thus expand the
81
application of plant RNA degradome data for the elucidation of post-transcriptional
82
gene regulation beyond small RNA-guided cleavage.
83
84
RESULTS
85
Signatures of Ribosome Footprints Were Observed in the RNA Degradome
86
Our previous analysis of plant RNA degradome data revealed positional enrichment
87
of 5′-truncated RNA ends in the proximity of motifs recognized by RNA-binding
88
proteins. This suggests that RNA-binding proteins attached to RNA may hinder RNA
89
degradation and result in protected RNA fragments (Hou et al., 2014). Therefore, we
90
suspected that the binding of ribosomes to mRNA may also protect mRNA from in vivo
91
degradation and leave ribosome footprints in the RNA degradome. To explore this
92
possibility, we first used PARE data to plot the positional distribution of 5′-truncated
93
mRNA ends in the junctions of the CDSs and UTRs. We predicted that, if PARE can
94
capture ribosome footprints in the same way as ribosome profiling (Ribo-Seq), which
95
delineates ribosome positions by generating ribosome-protected mRNA fragments
96
through in vitro nuclease digestion (Ingolia, 2010), we would observe a 3-nt periodicity
97
in the CDSs. A 3-nt periodicity reflects the stepwise movement of ribosomes during
98
active translation and has been reported in the analyses of Ribo-Seq data derived
99
from multiple species (Ingolia et al., 2009; Guo et al., 2010; Ingolia et al., 2011; Liu et
100
al., 2013; Bazzini et al., 2014; Juntawong et al., 2014; Vasquez et al., 2014).
101
Consistent with our prediction, 5′ ends of truncated mRNA (PARE data) generated
102
from Arabidopsis seedlings and inflorescences show strong 3-nt phasing in the 3′
103
terminus of the CDSs but not in the proximal region of the 3′ UTR (Figure 1A). A 3-nt
104
periodicity in the CDS region was also observed in the CDSs for PARE reads
5
105
generated from rice (Oryza sativa) inflorescences and soybean (Glycine max) seeds
106
(Figure 1A). Besides this phasing pattern, the three species all show preferential
107
accumulation of PARE reads in the translational frame (frame 1) of annotated CDSs
108
(Figure 1B). Although the enrichment in the translational frame is relatively small in
109
PARE data compared to that in yeast Ribo-Seq data reported previously (Ingolia et al.,
110
2009), the proportion of PARE reads falling in the translational frame is significantly
111
higher than that in the other two frames in the three replicates of Arabidopsis
112
inflorescence PARE data (Supplemental Figure 1). Similar to the previous finding in
113
the analysis of Ribo-Seq data (Liu et al., 2013; Bazzini et al., 2014; Juntawong et al.,
114
2014), PARE data of these three plant species also showed an evident increase in the
115
number of reads at positions 16 and 17 nt upstream of stop codons, a pattern
116
consistent with the deceleration of ribosome movement during translational
117
termination (Figure 1A). These common features shared between PARE data and
118
Ribo-Seq data strongly suggest both the presence of in vivo ribosome-protected
119
mRNA fragments in the plant RNA degradome and the occurrence of co-translational
120
RNA decay in plants.
121
122
Regular and Conserved RNA Degradation Patterns Were Found in Conserved
123
Peptide uORFs
124
Several uORFs in fungi, plants, and animals that encode conserved peptides are able
125
to block ribosomes at stop codons (Wang and Sachs, 1997; Raney et al., 2000; Gaba
126
et al., 2001; Hayden and Jorgensen, 2007; Hood et al., 2007; Uchiyama-Kadokura et
127
al., 2014). Among them, the conserved peptide uORFs (CPuORFs) of yeast CPA1 and
128
an Arabidopsis gene producing S-ADENOSYLMETHIONINE DECARBOXYLASE
129
(SAMDC/AdoMetDC1) have been demonstrated to induce mRNA decay through the
130
NMD pathway (Gaba et al., 2005; Uchiyama-Kadokura et al., 2014). To provide
6
131
additional evidence that the RNA degradome contains ribosome footprints, we
132
examined the positional distribution of 5′-truncated mRNA ends (PARE reads) derived
133
from several CPuORFs of Arabidopsis. In SAMDC, the position 16 nt upstream of the
134
uORF stop codon shows a predominant accumulation of PARE reads derived from
135
seedlings but only a weak enrichment of PARE reads derived from inflorescences
136
(Supplemental Figure 2). Besides SAMDC, we also examined PARE reads that
137
mapped to the CPuORFs in a small group of bZIP genes which regulate the translation
138
of downstream ORFs in response to sucrose concentration (Wiese et al., 2004).
139
Interestingly, these bZIP CPuORFs possess a ladder of PARE peaks at intervals of
140
about 30 nt, which is the size of a ribosome-protected fragment in Arabidopsis (Liu et
141
al., 2013; Juntawong et al., 2014) (Figure 2A). Counting from the 3′ end of bZIP
142
CPuORFs, the first and second PARE peaks are positioned about 16 and 46 nt
143
upstream of uORF stop codons, with a few reads present in the 30-nt window between
144
these two peaks. A third PARE peak at position -76 that was an additional 30-nt
145
upstream was observed in bZIP2 and bZIP11 CPuORFs (Figure 2A). The 30-nt
146
phasing of PARE peaks in the 3′ end of CPuORFs provides strong evidence to support
147
the notion that PARE captures the degradation fragments protected by an array of
148
stacked ribosomes. These three PARE peaks likely delineate the 5′ ends of
149
degradation fragments protected by one, two, or three adjacent ribosomes stalled at
150
a CPuORF stop codon, respectively. Notably, the 30-nt phasing was not evident in
151
these bZIP genes when we analyzed in vitro ribosome-protected mRNA fragments of
152
two Ribo-Seq data sets that were generated by independent groups (Liu et al., 2013;
153
Juntawong et al., 2014) (Figure 2A; Supplemental Figure 3).
154
Because bZIP CPuORFs were reported to repress the translation of the
155
downstream ORF under high sucrose concentration (Wiese et al., 2004), we used
156
modified RNA ligase-mediated rapid amplification of cDNA ends (RLM 5′ RACE) to
7
157
test whether treatment with 6% sucrose would affect the accumulation of degradation
158
fragments truncated at these bZIP CPuORFs. Indeed, a larger number of 5′ ends of
159
degradation intermediates corresponding to the first or second peak in bZIP2 and
160
bZIP11 CPuORFs were detected in Arabidopsis seedlings treated with 6% sucrose
161
compared to the untreated control plants (Figure 2B). This implies either an increase
162
in ribosomes arrested at these two uORFs or the enhancement of RNA degradation.
163
Taken together, these results indicate that degradation fragments appear to reflect the
164
dynamics of ribosomes on uORFs.
165
Although 64 CPuORFs (Hayden and Jorgensen, 2007) have been identified in
166
Arabidopsis by sequence comparison and annotated in The Arabidopsis Information
167
Resource database (TAIR10 annotation), most of them have not been demonstrated
168
to arrest ribosomes at specific positions. To further explore whether ribosome stalling
169
at stop codons is a common mechanism underlying the regulation mediated by
170
Arabidopsis CPuORFs, we globally analyzed the distribution of PARE reads mapped
171
to the 3′ end of the 64 Arabidopsis CPuORFs. Some Arabidopsis CPuORFs
172
predominantly accumulate PARE reads at position 16 nt upstream of stop codons and
173
some show an additional peak at position -45, -46 or -47 (Figure 3A). Besides bZIP
174
genes, CPuORFs in genes encoding several basic helix-loop-helix type transcription
175
factors, a trehalose-6-phosphate phosphatase, two methyltransferases and an
176
unknown protein were found to possess PARE peaks at these two specific sites
177
(Supplemental Figure 4), implying the stalling of ribosomes at these uORF stop
178
codons. The analysis of 35 rice CPuORFs showed the enrichment of PARE reads at
179
the same sites (positions -16 and -46) (Figure 3A), suggesting that ribosome stalling
180
at the stop codon of some CPuORFs is a conserved mechanism across species. To
181
know whether ribosome profiling could capture the signature of ribosome stacking in
182
CPuORFs as PARE, we also analyzed in vitro ribosome-protected mRNA fragments
8
183
of two Ribo-Seq data sets with the number of total reads greatly exceeding that of
184
PARE data sets we used (Liu et al., 2013; Juntawong et al., 2014). Intriguingly,
185
although position 16 or 17 nt upstream of the uORF stop codon shows an enrichment
186
of in vitro ribosome-protected mRNA fragments, the enrichment at these positions is
187
less prominent and does not accompany the enrichment at the positions near 30 nt
188
upstream (Figure 3B). The same analysis on genome-wide predicted uORFs having
189
lengths greater than 60 nt showed no preferential accumulation of PARE reads for
190
Arabidopsis or rice and Arabidopsis Ribo-Seq reads if predicted uORFs overlapping
191
CPuORFs were excluded (Figures 3A and 3B). Based on the analysis of PARE data
192
which harbor ribosome footprints, most Arabidopsis and rice uORFs may not cause
193
ribosome stalling at stop codons in the same way as many CPuORFs, at least under
194
the conditions and in the tissues that the PARE data were generated.
195
196
Regulatory uORFs Were Identified Using the Patterns of RNA Degradation
197
Fragments
198
We suspected that a few regulatory uORFs may not be conserved between
199
Arabidopsis and rice or that they might have been missed in the previous search
200
because of low sequence homology (Hayden and Jorgensen, 2007). Therefore,
201
analysis of the RNA degradome might provide an alternative approach for the
202
identification of regulatory uORFs that have the potential to stall ribosomes. We thus
203
reverse searched for uORFs using PARE reads peaking at the regions 16-17 and 45-
204
47 nt upstream of the stop codon of predicted uORFs. In addition to four CPuORFs
205
reported previously (Hayden and Jorgensen, 2007), we identified four Arabidopsis
206
uORFs with two predominant PARE peaks representing two tandem ribosomes
207
stacking at stop codons (Table 1 and Supplemental Figure 5). Further analyses were
208
then performed on these three novel uORFs to investigate sequence conservation and
9
209
their regulatory functions.
210
A 99-nt uORF in the 5′ UTR of CBL-INTERACTING PROTEIN KINASE 6 (CIPK6)
211
shows two PARE peaks at positions 16 and 46 nt upstream of the stop codon like
212
CPuORFs in bZIP genes (Figures 2 and 4A). We then compared the distribution of
213
reads obtained through Ribo-Seq and PARE on CIPK6. The Ribo-Seq data of light-
214
treated seedlings show a corresponding peak at position 16 nt but not 46 nt upstream
215
of the stop codon (Figure 4A). Moreover, position -17 accumulates more reads than
216
position -16 in Ribo-Seq data, whereas position -16 has the highest accumulation of
217
PARE reads. Predominant accumulation of ribosome-protected 5′ ends at positions 16
218
and 46 upstream of CIPK6 stop codon was not observed in the Ribo-Seq data of
219
normoxia (in air) seedlings (Supplemental Figure 6). Although the CIPK6 uORF was
220
not annotated in the TAIR database, it encodes a conserved peptide and has been
221
identified previously based on sequence homology by two groups (Takahashi et al.,
222
2012; Vaughn et al., 2012). Similarly, the conserved uORF in soybean CIPK6 also
223
possesses PARE peaks in 30-nt increments at the same positions relative to the uORF
224
stop codon (Figure 4B), implying that ribosome stalling might be a conserved
225
mechanism for CIPK6 uORF regulation.
226
The function of Arabidopsis CIPK6 uORF in repressing downstream ORF
227
expression was validated via transient expression assays by Ebina et al. (2015). We
228
confirmed the regulatory function of Arabidopsis CIPK6 uORF by generating stable
229
transgenic lines which harbored a reporter gene encoding β-glucuronidase (GUS)
230
driven by the Arabidopsis CIPK6 promoter containing a wild-type (WT) 5' UTR or a
231
uORF-deleted (ΔuORF) 5' UTR in which the start codon was converted into a stop
232
codon (Figure 4C). Overall, the ΔuORF transgenic lines showed a higher level of
233
expression of the reporter gene in most tissues (Figure 4C; Supplemental Figure 7),
234
indicating that the regulation mediated by CIPK6 uORF is widespread even under
10
235
normal growth conditions. Because an approximate 6-fold difference (P = 0.02, two-
236
tailed student’s t-test) was detected in the comparison of GUS activity, but the change
237
of GUS mRNA level was less than 2-fold and not statistically significant (Figures 4D
238
and 4E), CIPK6 uORF likely controls downstream ORF expression mainly at the
239
translational level.
240
The other two candidates of ribosome stalling uORFs we identified are located in
241
MYB34 and MYB51, which belong to the same clade of MYB transcription factors
242
involved in the regulation of glucosinolate biosynthesis (Celenza et al., 2005;
243
Gigolashvili et al., 2007; Frerigmann and Gigolashvili, 2014). Although MYB34 uORF
244
is longer than MYB51 uORF, they both possess two predominant PARE peaks at
245
positions 16 and 46 nt upstream of uORF stop codons (Figure 5A). The translation of
246
these two MYB uORFs is supported by the higher density of Ribo-Seq reads in the
247
predicted ORFs compared to that in the flanking regions (Liu et al., 2013) (Figure 5A).
248
However, the analysis of in vitro ribosome-protected mRNA fragments on these two
249
MYB uORFs with two Ribo-Seq data sets (Liu et al., 2013; Juntawong et al., 2014)
250
showed no preferential accumulation at these two sites (Figure 5A; Supplemental
251
Figure 6). The peptide sequences encoded by these two uORFs are highly conserved
252
at the C-terminus within the mustard (Brassicaceae) family (Figure 5B). Glucosinolates
253
are sulfur- and nitrogen-containing secondary metabolites that are found mainly in
254
plant species in the order of Brassicales (Grubb and Abel, 2006), explaining the
255
absence of conserved uORFs in many other plant species. The negative impact of
256
MYB34 CPuORF on the mRNA level of MYB34 was demonstrated previously in a
257
study of a mutant containing a PTC in this uORF (Bender and Fink, 1998). We
258
confirmed the negative regulation of MYB34 uORF on the expression of the
259
downstream reporter gene by converting the start codon into a stop codon in transient
260
expression assays (Figure 5C). The abolition of MYB51 uORF slightly increased the
11
261
expression of the reporter gene, but the change was not statistically significant under
262
the conditions we used for transient expression assays (Figure 5C). The identification
263
of MYB34 and MYB51 uORFs validates the use of RNA degradome data in
264
discovering lineage or species specific regulatory uORFs.
265
266
Ribosome Stacking Was Predicted in CDSs Using the RNA Degradome Data
267
Besides CPuORFs, the nascent peptide encoded by a CDS region in the first exon of
268
Arabidopsis CGS1, known as the MTO1 region, has been reported to block ribosome
269
elongation and induce RNA decay in response to S-adenosyl-L-methionine (AdoMet)
270
(Onouchi et al., 2005). Similar to the degradation fragments we observed in bZIP
271
CPuORFs (Figure 2), Haraguchi et al. (2008) detected a ladder of truncated 5′ termini
272
separated in length by approximately 30-nt increments in the MTO1 region together
273
with the 5′ upstream region after treatment with AdoMet. Moreover, the previous study
274
demonstrated that the truncated mRNA ends are defined by the 5′ edges of stalled
275
ribosomes in an array. Therefore, we assumed that the RNA degradome could also be
276
used in the identification of ribosome stacking occurring in the CDS region. To validate
277
this idea, we first compared the PARE peaks around the MTO1 region with the 5′ ends
278
of degradation intermediates reported previously (Yamashita et al., 2014). Although
279
the plants we used for PARE library construction were grown in soil without AdoMet
280
treatment, the major PARE peaks were in close proximity to the 5′ termini of
281
degradation intermediates reported previously (Supplemental Figure 8).
282
Next, we performed phasing analysis on PARE peaks in the CDSs for intervals from
283
20 to 40 nt. In the analysis of Arabidopsis seedling PARE data, the number of phased
284
regions decreased when the length of intervals increased (Supplemental Figure 9A).
285
However, the numbers of phased regions identified at intervals of 32 to 40 nt and 28
286
nt were significantly lower than that at the interval of 30 nt in the inflorescence PARE
12
287
data (Supplemental Figure 9B). To eliminate the phased regions occurring by chance,
288
we discarded candidates identified only in a single inflorescence sample. This resulted
289
in four phased regions at the intervals of 29 and 30 nt and one to three
290
regions at the other intervals (Supplemental Figure 9C). Based on this result, we thus
291
discovered four Arabidopsis protein-coding genes possessing a region potentially
292
associated with protection signatures of stacked ribosomes (Figure 6). PARE peaks
293
with prominent phasing were identified in genes encoding plastidic type I signal
294
peptidase 2B (PLSP2B), a pentatricopeptide repeat protein, a RING/U-box
295
superfamily protein, and an unknown protein (Figure 6). However, Ribo-Seq peaks
296
separated in a 30-nt interval were not detected in these regions, although the position
297
of the most 3′ phased PARE peak identified in PLSP2B showed predominant
298
accumulation of Ribo-Seq reads (Liu et al., 2013; Juntawong et al., 2014) (Figure 6;
299
Supplemental Figure 10). Notably, the A sites of stalled ribosomes putatively
300
associated with these four regions were all predicted to fall in frame 2 but not in the
301
translational frame annotated in TAIR (frame 1) if we assumed that the distance from
302
the ribosome-protected 5′ end to the presumed A site was 17 nt according to the result
303
shown in Figure 1. Stalling in the non-translational frame may suggest that the last
304
pausing ribosome in the four CDS regions we identified is arrested during the step of
305
translocation but not decoding.
phased
306
307
Analysis of 5′-Truncated mRNA Ends Upstream of miRNA-Guided Cleavage
308
Sites
309
Plant miRNAs can guide cleavage in the middle of target sites, resulting in truncated
310
mRNA fragments (Rhoades et al., 2002). However, growing evidence suggests that
311
plant miRNA can repress target translation (Brodersen et al., 2008; Iwakawa and
312
Tomari, 2013; Li et al., 2013b; Li et al., 2013c; Liu et al., 2013), although the underlying
13
313
mechanism is not well characterized. Because our analysis of the RNA degradome
314
revealed the footprints of stalled ribosomes in uORFs and the CDSs, we predicted that
315
a similar analysis of miRNA target genes would be useful for elucidating whether the
316
binding of plant miRNAs can directly block the movement of ribosomes in planta.
317
Therefore, we investigated the distribution of PARE reads in a 55-nt region upstream
318
of putative miRNA-guided cleavage sites in Arabidopsis and rice. Unlike the
319
predominant accumulation of PARE reads at positions 16 and 46 nt upstream of uORF
320
stop codons (Figure 2), no position-specific enrichment was found in this region except
321
at the putative miRNA-guided cleavage sites (Figure 7). This result thus suggests that
322
the major mechanism by which plant miRNAs repress target translation may not be by
323
acting as physical barriers to hinder the movement of ribosomes.
324
325
Discovery of Potential Footprints of Ribosomes Hindered by AGO7 in
326
Arabidopsis TAS3
327
Surprisingly, three Arabidopsis TAS3 genes shared a highly similar but unusual pattern
328
of truncated 5′ RNA ends upstream of the non-cleavable target site of miR390 (Figure
329
8A). A PARE peak was located immediately or 4 nt upstream of the first base of non-
330
cleavable target sites of miR390. In the proximal region upstream of this peak, there
331
were three additional PARE peaks arranged at regular intervals of 28 to 30 nt. This
332
regular degradation pattern in TAS3 highly resembled that detected in bZIP CPuORFs
333
(Figure 2). Therefore, we assumed that the peak adjacent to the non-cleavable miRNA
334
site might be the footprint of AGO7, which loads miR390, whereas the upstream peaks
335
likely also represented ribosome footprints. Intriguingly, TAS3 was annotated to be
336
non-coding RNA because of lack of a long ORF and the production of conserved trans-
337
acting siRNAs (tasiRNAs) when targeted by miR390 (Allen et al., 2005).
338
To determine whether TAS3 might serve as a template for translation, we first
14
339
predicted ORFs in three Arabidopsis TAS3 genes. All three TAS3 genes have an ORF
340
that terminates 7 to 11 nt upstream of the non-cleavable site of miR390 (Figure 8A).
341
The coding nature of the predicted ORFs in three TAS3 genes was evaluated with
342
Ribo-Seq data published previously (Liu et al., 2013; Juntawong et al., 2014). The
343
predicted ORFs in TAS3a and TAS3b have relatively dense ribosome-protected
344
fragments compared to other regions, supporting the coding ability (Figure 8A;
345
Supplemental Figure 11). Moreover, the major peaks revealed by Ribo-Seq were close
346
to the predominant PARE peaks in TAS3a and TAS3b. This result suggested that
347
translated ORFs were present upstream of the non-cleavable target site of miR390
348
and 5′-truncated RNA ends mapped to this region were likely ribosome footprints. If
349
these PARE peaks represent the footprints of ribosomes hindered by miR390-AGO7
350
complex but not the cleavage remnants directed by tasiRNAs, they should disappear
351
in the ago7 but be sustained in the mutant of RNA-DEPENDENT RNA POLYMERASE
352
6 (RDR6), which acts downstream of AGO7 in the tasiRNA biogenesis pathway
353
(Mallory and Vaucheret, 2010). As predicted, the result of modified RLM 5′ RACE
354
clearly showed that degradation fragments with 5′ ends mapped to these positions
355
were barely detected in ago7 (Figure 8B). On the other hand, the amounts of these
356
degradation fragments were comparable between WT and rdr6 plants although both
357
ago7 and rdr6 showed increased amounts of TAS3a transcripts compared to the WT
358
and no tasiRNA production (Figures 8B, 8C, and 8D). The PARE data of WT, rdr6 and
359
ago7 inflorescences showed the same result as the modified RLM 5′ RACE assay. All
360
PARE peaks in the ORFs of three TAS3 genes vanished in ago7 except the one at
361
position -58 in TAS3b (Supplemental Figure 12). The result of mutant analysis,
362
therefore, indicates that the formation of these 5′-truncated RNA ends upstream of the
363
non-cleavable target site of miR390 depends on AGO7 but is independent of tasiRNA.
364
Taken together, these results imply that non-cleavable targeting of AGO7 may arrest
15
365
ribosomes as a road block in planta. Because the last major PARE peaks in the ORFs
366
in TAS3a, TAS3b and TAS3c are located 19, 14 and 16 nt upstream of the first
367
nucleotide of the stop codons respectively, the miR390-AGO7 complex appears to
368
arrest a ribosome in the step of elongation on TAS3a but in the step of termination on
369
TAS3b and TAS3c.
370
371
DISCUSSION
372
The RNA degradome is composed of degradation products of endoribonucleases and
373
XRNs acting through diverse pathways. Here, we not only demonstrate that ribosome-
374
protected mRNA ends are pervasive in the RNA degradome,but also demonstrate
375
exciting new applications of RNA degradome data in the study of post-transcriptional
376
gene regulation. Predominant accumulation of 5′-truncated mRNA ends in a 30-nt
377
interval likely represents an array of stacked ribosomes in the uORF, CDS, or the
378
upstream region of non-cleavable binding sites of AGO7 (Figure 9). Genome-wide
379
analysis of 5′ -truncated mRNA ends mapped to uORFs suggests that many CPuORFs
380
may repress downstream ORF expression by stalling ribosomes at CPuORF stop
381
codons, whereas the majority of predicted uORFs lack this ability (Figure 3). In addition,
382
the analysis of 5′-truncated mRNA ends occurring upstream of miRNA target sites
383
uncovered the signature of ribosome stacking in the non-cleavable target site of
384
miR390 but not other cleavable target sites (Figures 7 and 8). Novel regulatory uORFs
385
and CDS regions with potential to cause ribosome stalling were identified through the
386
analysis of the RNA degradome (Figures 4, 5, 6 and Table 1).
387
388
Comparison of Ribosome Footprints in RNA Degradome Data and Ribo-Seq
389
Data
390
Accumulation of degradation intermediates starting at the 5′ edge or the A site of
16
391
stalled ribosomes has been reported in bacteria, yeast, and Arabidopsis (Hayes and
392
Sauer, 2003; Sunohara et al., 2004; Doma and Parker, 2006; Haraguchi et al., 2008),
393
and it has been proposed that endoribonucleases are involved in the production of
394
these degradation fragments. However, a recent genome-wide survey of
395
monophosphorylated 5′ RNA ends in yeast showed that XRN1 is also involved in the
396
accumulation of 5′-truncated RNA ends protected by ribosomes during RNA decay
397
(Pelechano et al., 2015). Using the plant degradome data, we identified an array of 5′-
398
truncated RNA ends separated at 30-nt intervals in uORFs and CDSs, implying an
399
array of stacked ribosomes (Figures 2, 4, 5, and 6). However, the 30-nt phasing pattern
400
was not observed in Ribo-Seq data for the regions we identified from RNA degradome
401
analysis. The discrepancy between Ribo-Seq data and RNA degradome data is likely
402
due to the different procedures of library preparation, although both methods could
403
capture ribosome-protected mRNA fragments. A typical Ribo-Seq protocol includes a
404
step to select fragments protected by a single ribosome but not stacked disomes or
405
trisomes (Ingolia, 2010). Therefore, a region associated with stacked ribosomes may
406
rather show a low density of protected fragments in Ribo-Seq data. On the other hand,
407
degradome sequencing, PARE, GMUCT or 5Pseq profiles truncated RNA termini
408
without monosome selection and can thus capture fragments protected by stacked
409
ribosomes. In addition, in the absence of monosome selection and translation inhibitor
410
treatment to block ribosomes on mRNA, these methods may also allow the
411
identification of fragments protected by ribosomes on distinct translation states.
412
A previous global study of yeast ribosome footprints demonstrated that ribosome-
413
protected fragments fall into two major groups (28-30 nt long and 20-22 nt long) that
414
are stabilized by different translation inhibitors (Lareau et al., 2014). The size of the
415
protected fragments reflects the configuration and translation state of the ribosomes.
416
In the three Arabidopsis TAS3 genes, we detected PARE peaks at 24-30 nt
17
417
immediately upstream of the first base of non-cleavable miR390 target sites (Figure
418
8A). The shorter intervals between two PARE peaks may imply a ribosome pausing at
419
the translocation stage instead of the decoding stage, which should lead to a 30-nt
420
protected fragment in Arabidopsis. In addition to technical differences, the interplay
421
between ribosome stalling and RNA decay likely enhances the signal of stalled
422
ribosomes in RNA degradome data. By contrast, the signal of stalled ribosomes might
423
be embedded in the footprints of active ribosomes in Ribo-Seq data. In summary, the
424
use of degradome data for the identification of regions associated with stacked
425
ribosomes may outperform the use of Ribo-Seq data in some cases.
426
427
Investigation of Regulatory uORFs using the RNA Degradome
428
Comprehensive identification of regulatory uORFs has been challenging as predicted
429
uORFs may not be translated or regulate downstream ORF expression through
430
distinct mechanisms (Barbosa et al., 2013). Prior to the development of Ribo-Seq,
431
translated uORFs with regulatory functions were often identified by mutant screening
432
or evolutionary conservation (Hill and Morris, 1993; Delbecq et al., 1994; Wiese et al.,
433
2004; Imai et al., 2006; Hayden and Jorgensen, 2007). Although Ribo-Seq data have
434
been used in the identification of translated uORFs (Fritsch et al., 2012; Liu et al., 2013;
435
Ingolia et al., 2014), the application of Ribo-Seq data to the identification of ribosome
436
stalling uORFs has not been reported. In this study, we show that some CPuORFs
437
over-accumulate 5′-truncated RNA ends with a signature of stacked ribosomes (Figure
438
2A). We further demonstrate that the accumulation of 5′-truncated RNA ends in bZIP
439
CPuORFs is enhanced in response to a high concentration of sucrose (Figure 2B).
440
The data thus support the previous hypothesis that the conserved peptides encoded
441
by these bZIP CPuORFs can stall ribosomes at stop codons in response to sucrose
442
concentration (Wiese et al., 2004), and suggest the application of degradome data in
18
443
the study of regulatory uORFs. Through the global analysis of 5′-truncated RNA ends
444
occurring in predicted uORFs, we identified novel ribosome stalling uORFs in
445
Arabidopsis. Notably, uORFs in MYB34 and MYB51 are conserved and specific in the
446
Brassicaceae family (Figure 5B). Since RNA degradome data of diverse plant species
447
are available in the public domain, these data sets might be very useful for the
448
identification of lineage or species specific ribosome stalling uORFs as evolutionary
449
conservation is not required for this type of analysis.
450
451
Analysis of the RNA Degradome Allows Dissection of Ribosome Pausing
452
Besides uORFs, CDS regions also possess 30-nt phased 5′-truncated mRNA ends
453
(Figure 6). Some regions likely encode nascent peptides which can stall ribosomes
454
during translation elongation as the MTO1 region in CGS1 or are upstream of
455
structured regions which can block the movement of ribosomes (Onouchi et al., 2005).
456
The one with the last peak of 5′-truncated RNA ends located 16 nt upstream of main
457
ORF stop codons as in the degradation pattern observed in CPuORFs may cause
458
ribosome pausing during translation termination. A previous study showed that the
459
binding of EUKARYOTIC RELEASE FACTOR 1 (eRF1) to three types of stop codons
460
causes a conformational change in the ribosome and a 2-nt shift toward the 3′ end of
461
mRNA (Kryuchkova et al., 2013). Because the position 17 nt upstream of the main
462
ORF stop codon is in frame with the 3-nt periodicity observed in the CDSs (Figure 1A),
463
the specific accumulation of 5′-truncated mRNA ends at this position suggests the
464
pausing of a ribosome with the A site at the stop codon. On the other hand, the 5′-
465
truncated mRNA ends peak 16 nt upstream of the stop codon may imply the stalling
466
of ribosomes after the conformational change of ribosomes induced by the binding of
467
eRF1. The integration of the RNA degradome data, protein sequences and RNA
468
structures will bring new insights into the regulation of ribosome pausing.
19
469
Exploration of Plant miRNA-mediated Translational Repression with RNA
470
Degradome
471
Unlike animal miRNAs, which mainly target the 3′ UTR, the majority of plant miRNAs
472
bind to their target CDSs through nearly perfect base pairing. Although cleavage sites
473
in the middle of plant miRNA target sites have been extensively validated by
474
degradome data in many species, translational repression mediated by plant miRNAs
475
has also been demonstrated through multiple approaches. Previous analysis of
476
Arabidopsis Ribo-Seq data showed that miRNA targets have lower translational
477
efficiency compared to non-miRNA targets (Liu et al., 2013). However, no preferential
478
accumulation of ribosome footprints was observed in the region upstream of miRNA
479
target sites in Arabidopsis Ribo-Seq data (Liu et al., 2013). Because Ribo-Seq has
480
some drawbacks in detecting stacked ribosomes or ribosomes pausing at the
481
translocation stage as we have discussed, we used RNA degradome data to
482
reexamine whether plant miRNAs can directly stall ribosomes . Consistent with the
483
previous observation in Ribo-Seq data (Liu et al., 2013), no site-specific enrichment of
484
5′-truncated RNA ends was found in the region upstream of Arabidopsis and rice
485
miRNA target sites (Figure 7). This strengthens the notion that directly blocking
486
ribosome movement through the binding of RISC contributes little to plant miRNA-
487
mediated translational repression in planta. Nevertheless, the binding of AGO7 to a
488
well characterized non-cleavable target site of miR390 seems to hinder the movement
489
of ribosomes and cause ribosome stacking as the upstream regions show signatures
490
of stacked ribosomes in the same way as bZIP CPuORFs (Figures 2 and 8). The same
491
degradation pattern corresponding to ribosome stacking among the three Arabidopsis
492
TAS3 genes suggests that this unique configuration of ORFs and non-cleavable
493
miR390 target sites might be crucial for tasiRNA production. Although the function of
494
ORFs upstream of the miR390 non-cleavable site has not been tested, positioning a
20
495
target site of miR173, another well-known tasiRNA trigger bound to AGO1 (Cuperus
496
et al., 2010), within 10 nt of a stop codon of an upstream ORF was shown to enhance
497
the production of artificial tasiRNA (Zhang et al., 2012). Moreover, a recently published
498
paper also demonstrated that an ORF surrounding the miR173 target site on TAS2 is
499
translated and plays a crucial role in tasiRNA production (Yoshikawa et al., 2016). That
500
signatures of stacked ribosomes exist close to the miR390 non-cleavable target site
501
revealed in this study strongly suggests that translation contributes to tasiRNA
502
biogenesis through a conserved mechanism regardless of miRNA triggers or AGO
503
involved.
504
A bottleneck in the development of RNA degradome data applications is the
505
complex composition of mRNA degradation intermediates, complicating interpretation.
506
The discovery of ribosome footprints in RNA degradome data opens up the possibility
507
of new applications of such data in post-transcriptional gene regulations beyond the
508
validation of miRNA-guided cleavage. It should be possible to apply the RNA
509
degradome data analyses demonstrated in this study to many other plant species that
510
have RNA degradome data available, enabling deeper insights into ribosome stalling
511
and mechanisms of RNA decay.
512
513
METHODS
514
Plant Materials and Growth Conditions
515
Arabidopsis thaliana (ecotype Col-0) used in this study was grown in soil or on 0.8%
516
Bacto-agar plates containing half-strength Murashige and Skoog (MS) medium (pH
517
5.7) and 1% sucrose under a 16/8-h light/dark cycle with an irradiance of 50-90 μmol
518
photons m–2 s–1 at 22°C. For the generation of PARE data, 11-d-old seedlings and
519
inflorescences of wild-type A. thaliana were used for total RNA isolation. For the
520
analysis of 5′-truncated RNA ends generated from bZIP2 and bZIP11 by the modified
21
521
RLM 5′ RACE assay, 10-d-old wild-type seedlings were transferred into liquid half-
522
strength MS medium with or without 6% sucrose. After incubation in growth chambers
523
with rotary shaking at 40 rpm under constant light for 24 h, the seedlings were collected
524
for RNA extraction. The inflorescences of Arabidopsis wild type, ago7 (GK-824A08-
525
025510), and rdr6 (CS24285) were harvested for the analysis of 5′-truncated RNA
526
ends generated from TAS3a with a modified RLM 5′ RACE assay and for PARE library
527
construction.
528
529
PARE Library Construction and Sequencing
530
Total RNA isolated by PureLink Plant RNA Reagent (Thermo Fisher) and MaxTract
531
high-density gel tubes (Qiagen) was used for PARE library construction following the
532
protocol published previously (Zhai et al., 2014). PARE libraries were constructed with
533
about 80 μg total RNA and then sequenced on the Illumina HiSeq 2500 platform.
534
535
Modified RLM 5′ RACE Assay
536
Modified RLM 5′ RACE assay was performed to detect 5′-truncated RNA ends using
537
GeneRacer Kit (Thermo Fisher). First, 2-3 μg of total RNA isolated by PureLink Plant
538
RNA Reagent and MaxTract high-density gel tubes was ligated with the 5′ RNA adapter
539
and then reversely transcribed with the oligo-dT primer. Next, cDNA was used as the
540
template for PCR analysis with a GeneRacer 5′ primer and a gene-specific primer.
541
Nested PCR was performed with a GeneRacer 5′ nested primer and a gene-specific
542
nested primer if no PCR products were detected in the primary PCR. Amplified
543
products of expected size were gel purified, cloned into pJET1.2/blunt Cloning Vector
544
or pCR4-TOPO TA Vector (Thermo Fisher) and sequenced. A target of miR159,
545
MYB65, was included as the positive control for the modified RLM 5′ RACE assay. The
546
primers are listed in Supplemental Table 1.
22
547
Analysis of 5′-truncated RNA End Distribution
548
In addition to the in-house PARE data of Arabidopsis, previously published PARE data
549
of Arabidopsis, rice, and soybean downloaded from the Gene Expression Omnibus
550
(GEO) (http://www.ncbi.nlm.nih.gov/geo/) were also analyzed in this study. Accession
551
numbers are given at the end of Methods. Trimmed reads were mapped to the
552
corresponding
553
(https://www.arabidopsis.org/; TAIR 10), the MSU Rice Genome Annotation Project
554
(http://rice.plantbiology.msu.edu/;
555
(https://phytozome.jgi.doe.gov/; Phytozome v11.0 : Gmax_275_Wm82.a2.v1 ) with
556
Bowtie (http://bowtie-bio.sourceforge.net/; v1.0.0). Only perfectly matched 20-nt
557
sequences were used in the following metagene analyses and sequences of low
558
complexity (repeats ≥ 15 nt) or with ≥ 10 hits in the genome were excluded. The
559
abundance of PARE sequences was assigned to the position corresponding to the first
560
nucleotide of the sequence. Known Arabidopsis and rice CPuORF and miRNA target
561
sites were retrieved from previous reports (Hayden and Jorgensen, 2007; Zheng et al.,
562
2012).
genomes
or
gene
sequences
Release
downloaded
6.1),
and
from
TAIR
Phytozome
563
For metagene analysis of PARE reads in the regions flanking the start codon and
564
the stop codon of annotated CDSs, the abundance of PARE reads at each position in
565
the defined regions on a transcript was first normalized by dividing the value by the
566
sum of PARE reads starting in the defined region. Then the relative abundance at each
567
position across the defined region was calculated as the sum of normalized
568
abundance of PARE reads starting at the same position for all genes. For heat maps
569
of PARE read distribution in regions upstream of uORF stop codons or miRNA guided
570
cleavage sites, we normalized the abundance of PARE reads at each position by
571
dividing the value by the sum of PARE reads starting in a 31-nt window flanking the
572
indicated position. The distribution of normalized PARE abundance was then clustered
23
573
using Ward’s method with R package (https://www.r-project.org/; version 2.15.2) and
574
displayed as heat maps.
575
576
Ribo-Seq Data Analysis
577
Previously published Ribo-Seq data sets of Arabidopsis were downloaded from the
578
GEO database. Accession numbers are given at the end of Methods. Trimmed reads
579
of length ≥ 20 nt were mapped to gene sequences downloaded from TAIR with Bowtie.
580
Two-nt mismatches were allowed for mapping Ribo-Seq reads of normoxia seedlings,
581
whereas perfect matches were used in the mapping of Ribo-Seq reads of light-treated
582
seedlings. The abundance of Ribo-Seq sequences was assigned to the position
583
corresponding to the first nucleotide of the sequence.
584
585
Identification of uORFs with Footprints of Stacked Ribosomes
586
RNA ends protected by monosome and disome pausing at uORF stop codons should
587
result in PARE peaks at positions 16 and 46 nt upstream of the stop codon. A custom
588
Perl script was used to identify uORFs possessing this degradation signature with in-
589
house Arabidopsis PARE data (Supplemental Script 1). First, the sequences of
590
Arabidopsis 5′ UTRs based on TAIR10 annotation were used to predict uORFs by
591
looking for an ATG codon paired with the nearest in-frame stop codon. Next, PARE
592
reads were mapped to the predicted uORFs by this Perl script. Candidates of uORFs
593
with a signature of ribosome stacking at the stop codon were selected based on the
594
following criteria. To capture the signature of ribosome stacking, the distribution of
595
PARE reads was evaluated in two 31-nt regions upstream of the stop codon. The first
596
region spans positions -1 to -31 and the second region spans positions -32 to -62, with
597
the first nucleotide of the uORF stop codon is set to 0. In the first region, the most
598
abundant peak (the first major peak) was required to be at position -16 or -17 and with
24
599
the number of raw reads ≥3. In the second region, the most abundant peak (the second
600
major peak) was required to be located at position -45, -46 or -47. Moreover, the
601
abundance of the major peaks was required to be at least 2-fold higher than that of
602
the second most abundant peak in the same region but outside the possible positions
603
for major peaks.
604
605
Identification of Degradation Signatures Representing Stacked Ribosomes in
606
the CDSs
607
To identify degradation signatures representing stacked ribosomes in the CDSs, we
608
first mapped trimmed PARE sequences to Arabidopsis CDSs based on TAIR10
609
annotation with Bowtie Because the protected degradation termini caused by three
610
stacked ribosomes are three predominant PARE peaks separated by about 30 nt, we
611
calculated two values, Peak_Abundance and Peak_Index, to evaluate the
612
predominance of PARE reads accumulated at each position. Peak_Abundance was
613
defined as the sum of PARE reads starting at the indicated position together with the
614
positions 1 nt upstream and downstream. Peak_Index was calculated by dividing the
615
Peak_Abundance by the total PARE reads starting in a 31-nt window flanking the
616
indicated position. Positions with Peak_Index ≥ 0.3 were selected as the first major
617
peak and were reset to 0 in further analysis. The second and third major peaks were
618
required to be at the regions between -29 and -31, and -59 and -61 with Peak_Index
619
≥ 0.3. In addition, the abundance of three major peaks had to be the highest in the
620
region for the calculation of Peak_Index and with raw reads ≥ 3. The abundance of the
621
three major peaks also needed to be 2-fold higher than that of the second most
622
abundant peak falling in the 31-nt window but outside the possible positions for major
623
peaks. To ensure that the degradation signature caused by stacked ribosomes was
624
prominent among all degradation events within a gene, a region was selected only if
25
625
all three major peaks were ranked in the top 1% of all positions with regard to PARE
626
abundance. To eliminate the phased regions occurring by chance, the candidates
627
uncovered from only a single sample were removed. The in-house Perl script for PARE
628
peak analysis in CDSs is provided as Supplemental Script 2. The same criteria were
629
applied for the phasing analysis of PARE peaks for intervals from 20 to 40 nt.
630
631
Generation of Transgenic Lines
632
A 1.2 kb DNA fragment of Arabidopsis CIPK6 promoter region plus 5′ UTR was cloned
633
into gateway vector pHGWFS7 upstream a GUS reporter gene through Gateway LR
634
Clonase II Enzyme mix (Thermo Fisher). Site-directed mutagenesis was applied to
635
change the start codon of CIPK6 CPuORF to a stop codon. The two constructs with
636
WT uORF and ΔuORF were transformed into wild-type Arabidopsis through the floral
637
dip method (Zhang et al., 2006). Primers used to clone the 1.2kb CIPK6 promoter
638
fragment are listed in Supplemental Table 1.
639
640
GUS Activity Assay and GUS Staining
641
The extract of ground tissues extracted by GUS extraction buffer (50 mM NaHPO4 pH
642
7.0, 10 mM 2-mercaptoethanol, 10 mM Na2EDTA, 0.1% sodium lauryl sarcosine and
643
0.1% Triton X-100) was mixed with MUG buffer (4-methylumbellifery β-D-glucuronide)
644
at 37°C for 20 min, and then the reaction was terminated by adding stop buffer (0.2 M
645
sodium carbonate). The fluorescence intensity of 4-methylumbelliferone (4MU) was
646
measured by fluorometer at 450 nm when excited at 365 nm. The amount of 4MU was
647
calculated with the standard curve and then normalized to the amount of total protein
648
which was measured using the Bradford dye-binding method with the BioRad Labs
649
protein assay kit. The GUS transgenic plants were stained in GUS staining solution
650
(0.1 M NaPO4 pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 1 mM K3Fe(CN)6 and 2 mM
26
651
X-Gluc) at 37°C overnight. After staining, samples were washed with 50% ethanol until
652
chlorophyll was removed, which took either overnight or several days depending on
653
the tissue. Then the stained samples were observed under a stereomicroscope
654
(SteREO Lumar.V12, ZESIS) and photographed with a digital camera (AxioCam MRc,
655
ZESIS).
656
657
Quantitative RT-PCR (qRT-PCR)
658
2 µg of total RNA of was used as a template for reverse transcription with ToolsQuant
659
II Fast RT kit (BIOTOOLS). The resulting cDNA was diluted 20-fold and 5 µL was used
660
for qRT-PCR in a 20 µL reaction with SYBR Green PCR Master Mix (Applied
661
Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems) using the
662
following program: 20 s at 95°C, followed by 40 cycles of 3 s at 95°C and 30 s at 60°C,
663
with an additional melt curve stage consisting of 15 s at 95°C, 1 min at 60°C, and 15
664
s at 95°C. TAS3a and GUS expression levels were normalized to the level of UBQ5
665
expression and were averaged from at least three independent biological samples,
666
followed by normalizing to the corresponding value of wild type. The primers used are
667
listed in Supplemental Table 1.
668
669
Protoplast Transient Assay
670
For construction of reporter plasmids, MYB34 and MYB51 5′ UTRs were amplified
671
from Arabidopsis genomic DNA and cloned into pJD301 between the cauliflower
672
mosaic virus 35S promoter and the firefly luciferase (LUC) coding region using the
673
NcoI restriction site. To abolish MYB34 and MYB51 uORFs, the start codon of uORFs
674
was converted into a stop codon by site-directed mutagenesis. Primers used in the
675
cloning of MYB34 and MYB51 5′ UTR sequences are listed in Supplemental Table 1.
676
Arabidopsis mesophyll protoplasts were isolated from 3 to 4-week-old rosette leaves
27
677
following the method described previously (Wu et al., 2009). Equal amounts (20 µg)
678
of reporter plasmids and internal control plasmids containing the 35S promoter driving
679
a GUS gene were cotransfected into 105 protoplasts in a PEG solution (40%
680
polyethylene glycol 4000, 0.2 M mannitol and 0.1 M CaCl2) at room temperature for 5-
681
10 min. The transfected protoplasts were incubated at 22°C in the dark for 16 h and
682
then lysed with Cell Culture Lysis Reagent (CCLR, Promega). LUC activity was
683
measured with a Luciferase Assay System (Promega) according to the manufacturer’s
684
instructions and normalized to GUS activity which was measured as described above.
685
686
RNA Gel Blot of Small RNA
687
Small RNA blot analysis was performed as described previously (Lee et al., 2015).
688
The probes used in the detection of small RNAs derived from TAS3 and U6 are listed
689
in Supplemental Table 1.
690
691
Alignment of Peptides Encoded by MYB34 and MYB51 uORFs
692
The sequences of MYB34 and MYB51 genes in different plant species were identified
693
by
694
(https://phytozome.jgi.doe.gov). The 5′ UTR sequences of orthologous genes
695
retrieved for uORF analysis with Serial Cloner (http://serialbasics.free.fr/; version 2.6)
696
are listed in Supplemental Data Set 1. Peptides encoded by MYB34 and MYB51 uORF
697
were then aligned with the use of Vector NTI software (Thermo Fisher).
a
BLASTP
search
and
downloaded
from
the
Phytozome
database
698
699
Accession Numbers
700
The PARE data generated in this study as well as public PARE data are available in
701
the GEO database under series GSE77549 (Arabidopsis in-house PARE data), and
702
accession numbers GSM280226 (public Arabidopsis inflorescence PARE data),
28
703
GSM647200 (public soybean seed PARE data), GSM434596 (public rice seedling
704
PARE data) and GSM476257 (public rice young inflorescence PARE data). The
705
Arabidopsis Ribo-Seq data sets used in this study are available in the GEO database
706
under
707
GSM1224475 (normoxia seedlings). Sequences of individual genes used in PARE
708
data analysis or functional assays and mutants used can be found in the TAIR or
709
Phytozome databases under the following accession numbers: AT3G02470 for Ath-
710
SAMDC, AT2G18160 for Ath-bZIP2, AT4G34590 for Ath-bZIP11, AT3G62420 for Ath-
711
bZIP53, AT4G30960 for Ath-CIPK6, Glyma09g14090 for Gly-CIPK6, AT5G60890 for
712
Ath-MYB34, AT1G18570 for Ath-MYB51, AT3G01120 for Ath-CGS1, AT3G17185 for
713
Ath-TAS3a, AT5G49615 for Ath-TAS3b, AT5G57735 for Ath-TAS3c, AT1G69440 for
714
Ath-ago7, and AT3G49500 for Ath-rdr6.
the
accession
numbers
GSM1226369
(light-treated
seedlings)
and
715
716
SUPPLEMENTAL DATA
717
Supplemental Figure 1. 5′-Truncated RNA Ends Show a 3-nt Periodicity and
718
Significant Frame Bias in the CDS.
719
Supplemental Figure 2. Overaccumulation of 5′-Truncated RNA Ends at the 3′ End
720
of Arabidopsis SAMDC uORF.
721
Supplemental Figure 3. The Comparison of PARE and Ribo-Seq Read Distribution
722
in Arabidopsis bZIP uORFs.
723
Supplemental Figure 4. Site-specific Enrichment of 5'-Truncated RNA Ends in
724
Arabidopsis CPuORFs.
725
Supplemental Figure 5. PARE Read Distribution in Arabidopsis uORFs with RNA
726
Degradation Signatures Representing Ribosome Stacking at Stop Codons.
727
Supplemental Figure 6. The Comparison of PARE and Ribo-Seq Read Distribution
728
in Arabidopsis CIPK6, MYB34 and MYB51 uORFs.
29
729
Supplemental Figure 7. Negative Regulation of CIPK6 uORF in Reporter Gene
730
Expression in Various Tissues.
731
Supplemental Figure 8. The Comparison of PARE Read Distribution with the 5′ Ends
732
of the Degradation Intermediates Previously Identified around the MTO1 Region of
733
CGS1.
734
Supplemental Figure 9. Phasing Analysis of PARE Peaks in the CDS.
735
Supplemental Figure 10. The Comparison of PARE and Ribo-Seq Read Distribution
736
in CDS Regions with 30-nt Phased PARE Peaks.
737
Supplemental Figure 11. The Comparison of PARE and Ribo-Seq Read Distribution
738
in Arabidopsis TAS3 Genes.
739
Supplemental Figure 12. Three Arabidopsis TAS3 Genes Accumulate AGO7-
740
dependent but RDR6-indepdennt Phased 5′-Truncated RNA Ends Upstream of Non-
741
Cleavable miR390 Target Sites.
742
Supplemental Table 1. Sequences of Primers for Cloning, Modified RLM 5′ RACE,
743
qRT-PCR and Probes for RNA Gel Blot Analysis.
744
Supplemental Data Set 1. Sequences for the Analysis of Conserved uORFs in
745
MYB34 and MYB51.
746
Supplemental Script 1. In-house Perl script for PARE peak analysis in uORF.
747
Supplemental Script 2. In-house Perl script for PARE peak analysis in CDSs.
748
749
ACKNOWLEDGEMENTS
750
We thank Shu-Hsing Wu and Tzyy-Jen Chiou of Academia Sinica for helpful
751
discussions and Ming-Che Shih for supporting Hsiao-Chun Chou. We also thank the
752
Academia Sinica Agricultural and Biotechnology Research Center core facilities for
753
help with transgenic plants and protoplast assays and Miranda Loney for English
754
editing of this paper. This work was supported by Academia Sinica.
30
755
AUTHOR CONTRIBUTIONS
756
H.-M.C. designed the research. C.-Y.H., H.-C.C. and H.-M.C. performed the
757
sequence data analyses. W.-C.L., H.-C.C., A.-P.C. and S.-J.C. carried out
758
experiments. C.-Y.H., W.-C.L. and H.-M.C. wrote the paper. All authors read and
759
approved the final manuscript.
Table 1. Arabidopsis uORFs with Two Predominant PARE Peaks 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
SD: seedlings; INF: inflorescences
31
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Figure 1. 5′-Truncated RNA Ends Show a 3-nt Periodicity and Frame Bias in the CDS.
(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 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 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 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.
(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.
Figure 2. bZIP uORFs Accumulate a Ladder of 5′-Truncated RNA Ends at 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 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 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.
(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 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 the graphs. TSS: transcriptional start site.
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 and rice PARE data
used in this analysis were published by German et al. (2008) and Li et al. (2010) respectively.
(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 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.
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 of annotated CPuORFs
and predicted uORFs (indicated in parentheses) and uORFs included in heat maps are shown above the heat
maps.
Figure 4. CIPK6 CPuORF Possesses Footprints of Stacked Ribosomes 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 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. (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 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 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. (C) Histochemical staining of randomly selected Arabidopsis T1 transgenic plants carrying a GUS reporter gene driven by
Arabidopsis CIPK6 promoter with wild-type (CIPK6pro:UTR(WT):GUS) and deleted uORF (CIPK6pro:UTR(ΔuORF):GUS).
The red arrowhead indicates the site mutated. Bar = 1 cm. (D) Comparison of GUS activity between WT and ΔuORF
transgenic plants. The amount of total protein was used for the normalization of GUS activity.
(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 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 (twotailed student’s t-test, n = 5).
Figure 5. Lineage-specific CPuORFs in MYB34 and MYB51 Generate 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 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 stop codon are
highlighted in red. TP50M, tags per 50 million.
(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
salsugineum. The alignment is colored according to residue conservation: red, identical residues; orange,
conserved residues; pink, block of similar residues.
(C) Transient expression assays of MYB34 and MYB51 uORF regulatory function using LUC reporter constructs
in protoplasts. The reporter constructs 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
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).
Figure 6. Analysis of PARE Data Reveals CDS Regions Potentially 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 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 highlighted in red with coordinates indicated above. TP50M tags per 50 million.
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 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 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 sites possessing PARE reads and included in heat maps are shown above heat maps.
Figure 8. Arabidopsis TAS3 Genes Accumulate AGO7-dependent but RDR6-independent Phased 5′-Truncated RNA
Ends Upstream of 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 Arabidopsis TAS3 genes. The PARE data of seedlings plotted were generated by this study and the RiboSeq 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 distances to the first nt of non-cleavable miR390
target sites. TP50M, tags per 50 million. (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 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
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 modified RLM 5′ RACE assay. In (A) and (B), the first nt of the non-cleavable miR390 target site is
assigned position 0. (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 < 0.01 (two-tailed student’s t-test, n = 4). (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 tasiRNA.
Figure 9. Schematic Representation of RNA Degradation Fragments 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.
(B) Degradation signatures caused by stacked ribosomes in the CDS. Position 0 is the 3′ edge of the most 3′
stalled ribosome.
(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 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 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.
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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 14, 2017
Supplemental Data
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