Plant Physiology Preview. Published on April 5, 2017, as DOI:10.1104/pp.17.00269
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Short title: Translation recovery after heat shock
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Corresponding authors:
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Rémy MERRET
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CNRS-LGDP UMR 5096, 58 av. Paul Alduy 66860 Perpignan, France
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Yee-yung CHARNG
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Agricultural Biotechnology Research Center, Academia Sinica, 128 Academia Rd. Sec. 2,
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Taipei, Taiwan 11529, ROC
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Article title:
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Heat-shock protein HSP101 affects the release of ribosomal protein mRNAs for recovery
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after heat shock
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Authors:
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Rémy MERRET1,2,3, Marie-Christine CARPENTIER2,3, Jean-Jacques FAVORY2,3, Claire
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PICART2,3, Julie DESCOMBIN2,3, Cécile BOUSQUET-ANTONELLI2,3, Pascal TILLARD4,
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Laurence LEJAY4, Jean-Marc DERAGON2,3,5, and Yee-yung CHARNG1
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Taipei, Taiwan 11529, ROC
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CNRS-LGDP UMR 5096, 58 av. Paul Alduy 66860 Perpignan, France
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Université de Perpignan Via Domitia, LGDP-UMR5096, 58 av. Paul Alduy, 66860
Agricultural Biotechnology Research Center, Academia Sinica, 128 Academia Rd. Sec. 2,
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Perpignan, France
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Intégrative des Plantes ‘Claude Grignon’, UMR CNRS/INRA/SupAgro/UM2, Place Viala,
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34060 Montpellier cedex, France
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Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie
Institut Universitaire de France
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One sentence summary:
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mRNAs coding for ribosomal proteins are preferentially stored during heat shock and released
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during recovery phase to enhance ribosome production in an HSP101-dependent manner.
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List of author contributions:
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RM performed all the experiments except confocal microscopy, bioinformatic analysis and
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elemental analysis. M-CC performed the bioinformatic analysis. CP and J-JF performed the
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confocal microscope analysis. JD performed the construction of transgenic lines. PT
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performed the elemental analysis under the supervision of LJ. RM, CB-A, J-MD, Y-YC
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designed the experiments. RM, J-MD and Y-YC wrote the paper.
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Funding:
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Research was supported Ministry of Science and Technology, Taiwan, ROC (MOST 103-
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2311-B-001-011-MY3 and 104-2923-B-001-001-MY3) to Y-YC and ANR RNP-bodies
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(ANR-2010 BLANC1707-01) and ANR Heat-Adapt (ANR-14-CE10-0015) to J-MD.
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Present address:
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RM is at CNRS-LGDP UMR 5096, 58 av. Paul Alduy 66860 Perpignan, France
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Email corresponding authors:
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[email protected]
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[email protected]
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Abstract (203 words)
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Heat shock (HS) is known to have a profound impact on gene expression at different levels,
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such as inhibition of protein synthesis, in which HS blocks translation initiation and induces
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the sequestration of mRNAs into stress granules (SGs) or P-bodies (PBs) for storage and/or
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decay. SGs prevent the degradation of the stored mRNAs, which can be re-engaged into
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translation in the recovery period. However, little is known on the mRNAs stored during the
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stress, how these mRNAs are released from SGs afterward, and what is the functional
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importance of this process. In this work, we report that Arabidopsis HEAT SHOCK
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PROTEIN101 (HSP101) knockout mutant (hsp101) presented a defect in translation recovery
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and SG dissociation after HS. Using RNA sequencing and RNA immunoprecipitation
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approaches, we show that mRNAs encoding ribosomal proteins (RPs) were preferentially
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stored during HS and that these mRNAs were released and translated in an HSP101-
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dependent manner during recovery. By 15N incorporation and polysome profile analyses, we
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observed that these released mRNAs contributed to the production of new ribosomes to
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enhance translation. We propose that, after HS, HSP101 is required for the efficient release of
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RP mRNAs from SGs resulting in a rapid restoration of the translation machinery by
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producing new RPs.
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Introduction
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Plants are often exposed to elevated temperature and have developed different layers of
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regulation to counteract adverse effects induced by heat stress. A well-defined plant response
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is the heat shock response (HSR) resulting in the production of molecular chaperones,
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including heat shock proteins (HSPs) (Kotak et al., 2007). The typical roles of these HSPs are
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to maintain the cellular proteostasis in limiting the production and accumulation of protein
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aggregates induced by HS and to confer thermotolerance (Kotak et al., 2007). Recently, the
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functions of HSPs in eukaryotes are extended to a wider scope. It appears that some of these
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proteins are also involved in the translational control of mRNAs under normal, stress
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conditions or recovery after stress (Walters and Parker, 2015).
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Especially, certain HSPs have been demonstrated to be involved in translation recovery after
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stress. Recently in Arabidopsis, small heat shock proteins (sHSPs) and HSP101 were shown
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to protect protein translation factors during HS (McLoughlin et al., 2016). sHSPs and HSP101
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are involved in the resolubilization of translation factors like eEF1B and eIF4A during the
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recovery phase. A similar mechanism was reported for human cells (Cuesta et al., 2000).
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During HS, HSP27 binds eIF4G and traps it in aggregates to prevent assembly of the cap-
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initiation complex and to rapidly enhance translation recovery. During recovery phase, these
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aggregates, called stress granules (SG), have to dissociate, and this mechanism implies also
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HSP proteins. In yeast, it was reported that HSP104 (the homologue of Arabidopsis HSP101),
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together with HSP70, is involved in disaggregation of SGs after HS (Cherkasov et al., 2013).
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Yeast hsp104 knockout mutant presents some defect in SG dissociation resulting in a slower
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translation recovery. In fact, the authors proposed that a slowest dissociation of SGs results in
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a longer release of translation factors trapped in SG, and thus limiting translation initiation
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recovery.
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The assembly of SG, in the same time with P-bodies (PB), is generated by a massive
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inhibition of translation induced by stress. This repression was observed under many stresses
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in yeast (Kuhn et al., 2001; Melamed et al., 2008), mammals (Hamilton et al., 2006; Liu et al.,
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2013) as well as in plants like Arabidopsis (Juntawong and Bailey-Serres, 2012), Medicago
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truncatula (Puckette et al., 2012), and rice (Park et al., 2012). HS is well known to repress
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translation inducing massive translation repression (Ueda et al., 2012; Merret et al., 2013).
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According to proteins composition, SG was attributed to mRNA storage while PBs are
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involved in mRNA decay. Although proteomic composition and assembly of these aggregates
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are increasingly documented (Buchan, 2014; Protter and Parker, 2016), little is known about
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the mRNAs stored in SGs, what is the functional role of the stored mRNAs during recovery,
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and how these mRNAs are released. Nonetheless, it is now well accepted that the dynamics of
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stress granules arise by an ATP-dependent mechanism and play a crucial role in mRNA
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metabolism (Jain et al., 2016).
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Recently, in Arabidopsis, formation and dissociation of SG were reported under hypoxia and
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reoxygenation (Sorenson and Bailey-Serres, 2014). The authors demonstrated the role of
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UBP1 in sequestration of poorly translated mRNAs during stress. They also showed that
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UBP1 granules are dissociated during reoxygenation. The authors proposed that UBP1
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orchestrates the translational control of mRNAs during and after stress. As in other
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organisms, SGs play an important role in plant stress tolerance and development, as mutants
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of SG components present some deficiency in acclimation to stress and/or development (Xu
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et al., 2006; Xu and Chua, 2009; Merret et al., 2013; Sorenson and Bailey-Serres, 2014;
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Perea-Resa et al., 2016)
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Nonetheless, few analyses address the involvement of the stored mRNAs in translation
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recovery. Here we show that the Arabidopsis hsp101 knockout mutant presents defects in SG
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dissociation and polysome recovery. We took advantage of this defect to identify mRNAs
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stored during HS and released at recovery. By RNA sequencing and RNA co-
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Immunoprecipitation (RIP) validation, we found that ribosomal protein (RP) mRNAs are
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preferentially stored during stress and released during recovery phase, the latter of which
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requires the chaperone function of HSP101. By
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demonstrated that new ribosomes can be produced independently of transcription, thus
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contributing to revive translation during the recovery period. We postulate that this
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mechanism could be a mean to rapidly adjust translational levels following successive
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stresses.
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N incorporation, we have further
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Results
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Polysome recovery is affected in hsp101 after HS
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To check translational activity in wild type compared to hsp101 (hot1-3, Lee et al.,
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2005), we analyzed polysome profiles on 7-day-old seedlings under the normal condition at
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22°C (control condition), after an HS treatment at 37°C for 1 h, and after 2 h of recovery at
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22°C following the HS (referred to as 2 h recovery hereafter, Fig. 1). Under the normal
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condition and during HS, no obvious differences were observed between wild type and
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hsp101 (Fig. 1A, D). After 1 h at 37°C, a massive polysome dissociation occurred that
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induces accumulation of monosomes (80S) and free 40S/60S ribosomal subunits (Fig. 1B, E).
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In wild type, after 2 h recovery, polysomes massively reappeared, suggesting a translational
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restart (Fig. 1C). Nonetheless, polysome recovery in hsp101 is affected compared to wild type
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(Fig. 1C, F). In fact, polysome increase is slower in hsp101, resulting in a higher amount of
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monosomes (compare the 80S peaks). To confirm this result, polysomes area was quantified
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for each condition using three biological replicates (Fig. 1G). A significant difference was
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only observed between wild type and hsp101 after 2 h recovery. HSP101 is a molecular
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chaperone protein; some missense mutants (dlt1-1 and dlt1-2) were shown to differentially
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affect its chaperone activity (Wu et al., 2013). dlt1-1 contains a single mutation C-to-T in
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HSP101 at position 599 near the N terminus of Nucleotide-Binding Domain2 causing the
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replacement of Thr by Ile. In dlt1-2, a single mutation C-to-T appears at position 33 in the N-
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terminal domain resulting in a His-to-Tyr substitution (Wu et al., 2013). Both maintained a
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normal level of HSP101 protein but dlt1-2 results in substantial loss of the chaperone function
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of HSP101 as dlt1-1 mutation as little effect on HSP101 chaperone activity. Moreover dlt1-2
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mutation induces thermotolerance defect, while dlt1-1 does not. Like hsp101, dlt1-2 seedlings
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are not able to survive under short-acquired thermotolerance (Wu et al., 2013). To check if
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HSP101 chaperone activity is involved in translation recovery, the polysome profiles of dlt1-1
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and dlt1-2 were analyzed after 2 h recovery (Supplemental Fig. S1). Only dlt1-2 presented
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defect in polysome recovery suggesting that HSP101 chaperone activity is involved in
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translation recovery. We tried to correlate this defect with a defect in growth rate after HS. To
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this end, the extension of root length was measured but no differences were observed between
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the wild type and hsp101 during recovery (data not shown).
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To check if this process is also correlated with a defect in SG dissociation, appearance
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and disappearance of SGs were monitored (Fig. 2 and Supplemental Fig. S2) with the
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Arabidopsis transgenic lines expressing the polyA binding protein 2 fused to the red
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fluorescent protein (PABP2-RFP) under the PABP2 promotor in the wild type or hsp101
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background. The levels of PABP2-RFP and HSP101 were checked by western blot
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(Supplemental Fig. S2A). SG accumulation and dissociation were analyzed on different roots
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by confocal microscope (Fig. 2 and Supplemental Fig. S2B). A defect in SG dissociation was
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observed in hsp101 compared to wild type after 2 h recovery (Fig. 2C vs. 2I, and
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Supplemental Fig. S2B), whereas no difference can be observed under normal and HS
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conditions (Fig. 2A vs. 2G and 2B vs. 2H). A SG quantification was performed in wild type
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and hsp101 to confirm this observation (Fig. 2M). According to these data, we proposed that
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HSP101 could be involved in translation recovery by regulating the dissociation of SG.
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Identification of stored mRNAs released in an HSP101-dependent manner
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Although the involvement of the yeast HSP104 in SG dissociation is known
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(Cherkasov et al., 2013), the nature of mRNA stored and released was not identified. As
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stored mRNAs could play a role in translation recovery, we designed an RNA sequencing
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strategy to identify them. Because SG purification could be difficult and quite challenging, we
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decided to take advantage of the hsp101 translation defect to identity these mRNAs with the
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idea that stored mRNAs will come back slower in translation in hsp101 compared to wild
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type. To do that, we performed RNA sequencing on total and polysome-associated mRNAs
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purified from wild type and hsp101 seedlings under the normal condition at 22°C, after 1 h at
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37°C, and after 2 h recovery (Supplemental Table S1). Each condition was performed on two
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biological repeats.
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Prior to analysis, for each condition, reads per kilobase per million mapped
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reads (RPKM) values obtained between replicates were compared. Each set of experiment
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was found to be reproducible as demonstrated by plotting read counts pairwise between
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replicates (with R2 ≈ 0.99) (Supplemental Table S1). Next, only transcripts with at least 1
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RPKM in 1 library were kept, resulting in 19,599 transcripts. The aim of the RNA sequencing
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is to identify mRNAs first stored during HS and next released in an HSP101-dependent
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manner. For stored mRNAs, we reasoned as follows: a stored mRNA must be stable and
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released from translation during HS, i.e. down regulated at polysomes level. To identify those
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mRNAs, a fold change (FC) analysis was performed at total RNA level between HS and
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control condition. The same analysis was also applied for polysomal RNA. A transcript
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having an FC below 0.5 was considered as down-regulated and was considered as stable with
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an FC between 0.5 and 2. To identify transcripts that were stored during HS (and not
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degraded as in Merret et al., 2015; Merret et al., 2013), we selected from the total RNA
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population heat “stable” mRNAs (having an FC between 0.5 and 2) that were clearly down
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represented (having an FC below 0.5) in the polysomal fraction after HS. According to the
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criteria, 11,558 and 11,779 transcripts are stable during HS in wild type and hsp101,
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respectively, with an overlap of 90% between both genotypes (Fig. 3A). Then, only common
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transcripts between wild type and hsp101 were kept, and their translation repression was
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analyzed. 3,309 transcripts were found to be downregulated at the polysomal level in both
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genotypes (Fig. 3A and Supplemental Table S2).
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To identify among the 3,309 transcripts, those released during recovery in an HSP101-
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dependent manner, we measured their translation efficiency (TE) (their ratio in polysomes vs.
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total RNA), during and after HS, in wild type and hsp101. As shown in Fig. 3B and 3C, TEs
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of these mRNAs were generally higher during recovery compared to HS, suggesting that most
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of the stored mRNAs during HS can come back rapidly in translation after stress. As hsp101
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presents a defect in polysome recovery, we decided to compare the TEs of these 3,309
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mRNAs during recovery in wild type and hsp101. To do that, a scatter plot was performed
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between wild type and hsp101 for the three conditions (Fig. 3D and Supplemental Fig. S3).
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Next, a linear regression was calculated for each condition with the 3,309 dots. Although their
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TEs are identical under control and HS conditions (Supplemental Fig. S3, linear regression
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close to 1), during recovery, TEs in hsp101 appeared to be affected compared to wild type as
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the linear regression decrease close to 0.8. Out of 3,309 transcripts, 2,103 showed TEs lower
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in hsp101 compared to wild type (red dots on Fig. 3D and Supplemental Table S3). Since SG
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dissociation was affected in hsp101, we propose that these 2,103 mRNAs are stored in SG
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during HS and released in an HSP101-dependent manner.
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mRNAs coding for RPs are stored during HS and released during recovery
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To go further in the functional role of the stored mRNAs during recovery, we
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performed a gene ontology (GO) analysis on the stored mRNAs released during recovery in
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an HSP101-dependent manner. Two molecular functions were enriched in the 2,103 mRNA
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population: “protein binding” (GO:0005515, p-value 0,000202) and “structural constituent of
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ribosome” (GO:0003735, p-value 6,81e-54) (Supplemental Fig. S4). The second GO term is
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highly enriched, and most of the mRNAs present in this group encode large or small subunits
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ribosomal proteins (RPL or RPS). According to our RNAseq database (Supplemental Table
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S1), 276 nuclear genes coding for RPS or RPL are expressed at seedlings stage. 219
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correspond to cytoplasmic RPs, 50 to chloroplastic RPs and 7 to mitochondrial RPs. Thus, to
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have a better overview of translation activity of mRNAs coding for RPs, TE was determined
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for all of them in the three conditions (control, HS, and recovery) in wild type and hsp101
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(Supplemental Table S4). TEs were clustered according to their behavior on three heatmaps
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(Fig. 4 and Supplemental Fig. S5). According to the clustering, a major group can be
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distinguished in the three class of RPs. This group is highly present for cytoplasmic RPs with
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179 members (81.7 %) compared to chloroplastic and mitochondrial RPs with 27 (54 %) and
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4 (57 %) members, respectively. This group has members with low TE during HS compared
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to the control, and their translation recovery seemed to be HSP101-dependent (Fig. 4 and
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Supplemental Fig. S5). We propose that members of this group are stored during HS and
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released directly or indirectly by HSP101 during the recovery phase. To confirm this
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hypothesis, we performed qPCR validation on two candidate transcripts of RPS23B
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(At5g02960) and RPS29B (At3g44010) (Fig. 5A, B). As expected, their TEs were
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significantly and identically reduced during HS in wild type and hsp101 whereas translation
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recovery was affected in hsp101 for both transcripts.
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To confirm that these mRNAs are stored in SGs during HS, a RIP approach coupled to
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RT-PCR was performed using a transgenic line expressing UBP1a fused to the green
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fluorescent protein (GFP). UBP1a was previously reported to localize in SG during stress
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(Weber et al., 2008; Sorenson and Bailey-Serres, 2014). The RIP experiment was performed
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under the normal condition at 22°C, after 1 h at 37°C, and after 2 h recovery. After checking
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immunoprecipitation efficiency (Fig. 6A), the association of RPS23B and RPS29B mRNAs
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with UBP1a was analyzed. RPL37B mRNAs was used as a positive control as this transcript
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was previously showed as an mRNA partner of UBP1 under hypoxia (Sorenson and Bailey-
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Serres, 2014). According to our data, this transcript seems to be stored during HS and
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released in an HSP101-dependent manner. For all transcripts, association with UBP1a was
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highly induced during HS (Fig. 6 B, C, D) and reduced or abolished during the recovery
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phase, coincident with the model where RP mRNAs are stored during HS and released during
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the recovery phase. Thus, we propose that HS induces a massive storage of mRNAs coding
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RPs and these mRNAs are released during recovery to enhance translation.
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Polysome recovery can be independent of transcription restart
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The storage of mRNA coding for RPs during HS suggests that the production of new
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ribosomes is a critical step to resume translation at recovery, meaning the new subunits have
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to be produced. To test this hypothesis, polysome profiles were done at different time points
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during recovery. We hypothesized that if pre-existing free ribosomal subunits (40S and 60S)
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can be reused during recovery, their amount will be decreased with the concomitant increase
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of 80S/polysomes. To test this hypothesis, the area of 40S/60S and 80S/polysomes were
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determined for each condition and normalized according to the respective area for HS
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condition. After 30 min recovery, monosomes (the 80S peak) decreased, and polysomes
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increased as compared to the HS situation, suggesting a translation restart (Fig. 7A).
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However, curiously, the amount of free ribosomal subunits did not change as compared to HS
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condition (ratio = 1.08 ± 0.03). After 1 h recovery, polysomes continued to increase and
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reached a maximum level while the amount of free ribosomal subunits remained, again,
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constant (ratio = 1.14 ± 0.09). After 2 h recovery, the amount of free ribosomal subunits
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decreased, but the polysomes stopped to increase. These observations strongly suggest that,
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up to 1 h recovery, pre-existing free ribosomal subunits could not fully account for the
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translation restart and that new ribosomal subunits must come into play with the synthesis of
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RPs.
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The production of new RPs could result from the translation of either de novo
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transcribed mRNAs or those released from SGs. To discriminate between these two
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possibilities, seedlings in the recovery phase were incubated on liquid medium supplemented
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or not with the transcription inhibitor, actinomycin D, to block both polymerase I and II
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activities (Bensaude, 2011). After 2 h recovery, polysome profiles were performed. As shown
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in Fig. 7B, although transcription is blocked, polysome recovery still took place, suggesting
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that a least a portion of new polysomes can be formed without transcription. To prove that
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new ribosomes can be formed in the time frame of the recovery period without necessarily
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mobilizing pre-existing, heat-dissociated ribosomal subunits, a
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N incorporation experiment
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was performed after stress. After
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amount was determined for proteins present in the polysomal fractions (essentially composed
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of ribosomes). These data were compared to a negative control (puromycin treatment) to be
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sure that incorporated
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shown in Fig. 7C, 15N was detected in polysomes after 2 h recovery, suggesting the formation
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of new ribosomes in this frame time. The same incorporation was performed with
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actinomycin D treatment (Fig. 7C). Although transcription is blocked, 15N incorporation was
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still detected in polysomes after 2 h recovery.
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N incorporation and polysome purification, the
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N signal only comes from proteins associated with polysomes. As
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As ribosome biogenesis needs mature rRNAs produced by RNA polymerase I, we
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investigated the effect of HS on nucleoli architecture. Nucleolus architecture is highly
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dependent on the production of a proper amount of rRNAs. One of the major components of
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the nucleolus is the dense fibrillar component (DFC) that consists of newly transcribed rRNA.
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Fibrillarin is a nucleolar protein known to localize to the DFC, forming ring-like structures in
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the nucleolus (Picart and Pontvianne, 2016) and can be used as a marker of nucleolus integrity
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(Pontvianne et al., 2013). Using FIB2-YFP stable line, the nucleolus status was checked under
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normal and HS conditions. As shown in Supplemental Fig. S6A and S6B, FIB2 formed ring-
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like structures both in normal and HS conditions, whereas actinomycin D treatment (that
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inhibits polymerase I activity) disrupts FIB2 localization (Supplemental Fig. 6C). Thus, HS
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did not affect nucleolus architecture, suggesting that RNA polymerase I activity was not
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significantly affected under this condition. All together, these data suggest that new
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ribosomes can be formed shortly after HS by mobilizing stored RP-coding mRNAs.
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Discussion
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Translation repression and storage of RP mRNAs during stress
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Although conditions leading to SG formation are now well documented, how specific
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mRNAs are stored and released from these aggregates, and the functional consequences of
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this process remain unclear. Here, using the hsp101 translation recovery defect, we observed
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that 210 of 276 nuclear mRNAs coding for RPs are preferentially stored under HS and
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released in the recovery period in an HSP101-dependent manner. Translation inhibition of RP
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mRNAs was already observed for different stresses like hypoxia (Branco-Price et al., 2008),
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sucrose deprivation (Nicolaï et al., 2006) or water deficit (Kawaguchi et al., 2004), suggesting
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that inhibition of ribosome biogenesis is a conserved stress response. This dampening of
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translation during stress was attributed to energy conservation (Branco-Price et al., 2008), as
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ribosome biogenesis is highly ATP consuming. Recently, it was reported that the association
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of RP mRNAs with UBP1 is enhanced under hypoxia (Sorenson and Bailey-Serres, 2014).
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Our data suggest that the same phenomenon occurs under HS (Fig. 6). Thus, it appears that
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RP mRNAs are translationally repressed and stored during stress. In yeast, the same
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observation was reported under glucose deprivation, while adding back glucose led to RP
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mRNAs translation recovery (Arribere et al., 2011). Interestingly, in this case, few genes can
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be reactivated for translation upon glucose replenishing, and most of them were attributed to
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ribosomal protein genes (Arribere et al., 2011). We observed the same mechanism in
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Arabidopsis under HS, suggesting a common signalling pathway in regulating RP mRNAs
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translation across eukaryotes. In yeast, it was suggested that hsp104 translational defect is due
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to a defect in the release of preinitiation complexes stored in SG (Cherkasov et al., 2013). Our
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data go further in this process and show that the nature of stored mRNAs could also influence
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the translation recovery.
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How to coordinate de novo ribosome synthesis after stress?
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We show in this work that new ribosomes can be formed shortly after HS by mobilizing
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stored RP-coding mRNAs. Ribosome biogenesis needs a fine-tuning between rRNA
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production mediated by RNA polymerase I and RP production mediated by RNA polymerase
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II. Therefore, an important question is how to coordinate the timely production of rRNAs and
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RPs to assemble new ribosomes shortly after a stress event. Previously it was reported that, in
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tomato, the production of rRNA primary transcripts is maintained under HS, while the
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maturation process is abolished, leading to the accumulation of primary transcripts in the
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nucleolus (Nover et al., 1986). The same observation was recently proposed also for yeast
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(Kos-braun et al., 2017). The authors showed that pre-rRNA continues to be synthesized upon
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stress and suggested that continued transcription of rDNA allows maintenance of nucleolar
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integrity to rapidly respond to environmental changes. This result agrees with our observation
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that the nucleolus architecture (a proxy of polymerase I activity) is maintained during HS in
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Arabidopsis (Supplemental Fig. S6A, B). The coordinated control during the stress of
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essential molecules needed to generate new ribosomes (primary rRNA production in the
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nucleolus and storage of RP-coding mRNAs in SGs) could be critical to synchronize
367
ribosome synthesis in the recovery period. This hypothesis is in agreement with our data
368
showing that RNA polymerase I and II activities are not needed to synthesize new ribosomes
369
early in the recovery period. The rapid restart of translation, only minutes after stress, could
370
therefore benefit from the HSP101-dependent release of RP-coding mRNAs from SGs, which
371
is to be synchronized with the maturation of primary rRNAs in the nucleolus, leading to new
372
ribosome formation.
373
374
Ribosomal subunits under stress and during recovery
375
The dynamics of ribosomal subunits we observed during and after the stress is supporting our
376
major conclusion that RP synthesis with stored mRNAs is needed to resume translation in the
377
recovery period. As shown in our polysome profiles (Fig. 7A), HS induces a massive
378
dissociation of polysomes and the accumulation of monosomes and free 40S and 60S
379
subunits. This is expected as the ribosomal subunits released following translation termination
380
cannot easily re-engage in new rounds of protein synthesis in a stress situation. Early in the
381
recovery period, one would expect that the re-activation of translation will increase the level
382
of polysomes with a corresponding decrease in monosomes and free subunits. However, this
383
is not what we observed as the level of free subunits was stable even 1 h in the recovery
384
period, when the polysome level is strongly increased. This phenomenon suggests that newly
385
synthetized ribosomal subunits, (and not only the pre-existing ones), are contributing to the
386
build up of polysome recovery.
387
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12
388
Why new ribosomes were produced instead of salvaging the pre-existing ones? One
389
possibility is that part of the pre-existing subunits have been damaged or irreversibly modified
390
by the stress and cannot be easily recycled. Indeed, under stress conditions that affect energy
391
status, alteration of ribosome subunits is one strategy that can preserve cellular energy
392
homeostasis. It was reported that under stress conditions or after translation inhibition,
393
modification of ribosomal subunits could occur through ubiquitination both for 40S and 60S
394
subunits (Kraft et al., 2008; Higgins et al., 2015). It was proposed that this modification not
395
only controls translational status under stress but also induces the ribosome degradation
396
pathway, called ribophagy (Kraft et al., 2008). To evade HS-inducing modifications, the
397
protein aggregation process was proposed to be a specific and reversible pathway to maintain
398
proteins activity for recovery phase (Wallace et al., 2015). This study suggests that free
399
ribosome subunits may not be able to efficiently aggregate under HS, making them more
400
sensitive to modification and/or degradation. This could be physiologically relevant as the
401
reduction in ribosome abundance was proposed to be a rapid and adaptive stress response
402
during proteotoxic stress in yeast (Guerra-Moreno et al., 2015). Ribosomal subunits
403
ubiquitination can occur rapidly upon stress (after 1 h DTT for human cells, Higgins et al.,
404
2015), whereas ribosomal subunits cleavage increase only significantly after 4 hours (Kraft et
405
al., 2008). Thus we postulate that the same mechanisms could occur during and after HS in
406
Arabidopsis and one way to recover from these effects would be to store RP mRNAs in SG to
407
enhance ribosome production during recovery phase.
408
409
Another challenging question is how RP mRNAs could be specifically targeted for this
410
massive sequestration and release process. A common cis-element “TAGGGTTT” was
411
previously identify on Arabidopsis RP mRNAs and was shown to be important in the context
412
of translation efficiency during photomorphogenesis (Liu et al., 2012). However, this cis-
413
element is also present on RP mRNAs that are apparently not stored and released following
414
HS. Therefore, it is unclear at the moment if this cis-element is involved or not and further
415
effort will be needed to determine the regulators, both cis and trans, required for sequestrate
416
and release of most RP mRNAs in a heat-stress situation.
417
418
In this study, we identified stress granule associated mRNAs using RNA sequencing
419
approach. According to our criteria, 210 of 276 nuclear RP mRNAs are stored during HS and
420
released in the recovery period in an HSP101-dependent manner (Supplemental Table S4).
421
This allows us to identify the functional role of stored RP mRNAs for recovery period.
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13
422
Moreover, another GO term was enriched in our analysis (“protein binding” GO:0005515).
423
As this term encompasses a large group of proteins with distinct functions, it is difficult to
424
draw a model around this term. Nonetheless, we cannot exclude that other mRNAs with
425
specific functions are stored during HS and also play a role during recovery.
426
427
Material Methods
428
429
Growth conditions
430
Analyses were carried out with 5- or 7-day-old whole plantlets grown on synthetic Murashige
431
and Skoog (MS½) medium (Duchefa) containing 1% sucrose and 0.8% plant agar at 22°C
432
under a 16h light/ 8h dark regime. For HS treatment, plates were immerged in a water bath at
433
37°C. For recovery, plates were transferred at 22°C under previous conditions.
434
435
Generation of transgenic plants
436
A vector containing pPABP2-tRFP-PABP2 was transformed into wild type (Col0) by floral
437
dip transformation. Next, a cross between this line and the hsp101 mutant (hot1-3, Lee et al.,
438
2005) was realized to obtain pPABP2-tRFP-PABP2 in the hsp101 background. The
439
expression level of RFP-PAB2 and HSP101 was confirmed by western blot analysis.
440
Transgenic lines with levels similar to wild type were kept for confocal analysis.
441
442
Polysome profile analysis and quantification
443
Polysome profiles were performed as described previously (Merret et al., 2013). Briefly, 400
444
mg of tissue powder were homogenised with 1.2 mL of lysis buffer (200 mM Tris-HCl pH
445
9.0, 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 1% detergent mix (1% Tween20, 1%
446
Triton, 1% Brij35, 1% Igepal), 1% sodium deoxycholate, 1% polyoxyethylene (10) tridecyl
447
ether, 5 mM DTT, 50 μg.mL-1 cycloheximide, 50 μg.mL-1 chloramphenicol, 1% protease
448
inhibitor cocktail, Sigma). Crude extract was incubated 10 min on ice. After centrifugation,
449
the supernatant was clarified with a 0.2 μM filter. 360 μL (= 120 mg) of crude extract was
450
loaded on a 15%-60% sucrose gradient (9 mL). Ultracentrifugation was performed with an
451
SW41 rotor at 38,000 rpm (≈ 180 000g) for 3 h. Polysome profile analyses were performed
452
with an ISCO absorbance detector at 254 nm. For normalization and direct comparison of
453
profiles, the same volume of crude extract was loaded on the sucrose gradient for each
454
condition. For quantification, the percentage of polysomes for each condition was determined
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
14
455
using Image J as the ratio between polysomes area and total area (including 40S, 60S,
456
monosomes and polysomes areas).
457
458
Confocal microscopy analysis and stress granules quantification
459
Confocal microscopy was performed on 5-day-old whole seedlings as described in Merret et
460
al., 2015. For SG monitoring, at least 5 different roots for each condition were analyzed. For
461
nucleoli analysis, FIB2-GFP line was used (Pontvianne et al., 2013). 25 nucleoli were
462
analyzed for each condition on 5 different seedlings. Stress granules were quantified using
463
plugins of Fidji (http://fiji.sc/, Schindelin et al., 2012) on 5 seedling roots per condition. First,
464
20 iterations of deconvolution were applied using Iterative Deconvolve 3D on 15-slide
465
(overlapping) stacks of 5.46 μm thick. Centre of granules were then counted using 3D Objects
466
Counter with a threshold of 1000, excluding signal from structures smaller than 50 voxels.
467
468
RNA sequencing
469
Total RNA extraction was performed by using RNeasy® Micro Kit (Qiagen) according to
470
manufacturer’s instructions. For polysomal RNA extraction, fractions corresponding to
471
polysomes were pooled. Two volumes of guanidium 8M and three volumes of absolute
472
ethanol were added. After overnight precipitation, centrifugation (16,000g, 45 min, 4°C) was
473
performed. Pellets were resuspended in RLT buffer (Qiagen). Next, RNA was extracted with
474
RNEasy® Micro Kit. RNA quality was assessed with Agilent 2100 bioanalyzer prior to RNA
475
sequencing. Libraries preparation was performed with TrueSeq Illumina kit. Libraries were
476
multiplexed and sequenced (PE 2x150) on an Illumina HiSeq 2500. For each library, at least
477
40 million reads were obtained. Reads filtering and mapping were performed as described in
478
Merret et al., 2015. For each condition, the mean value of RPKM (read per kilobase per
479
million mapped reads) was determined between replicates. Transcripts, which did not have at
480
least 1 RPKM in 1 condition, were removed from the analysis. FC was determined as the ratio
481
between transcript quantity during HS and normal condition or during recovery and normal
482
condition. TE was determined as the ratio between transcript quantity in polysomal RNA and
483
total RNA. Raw data are reported in Table S1.
484
485
GO and heatmap analysis
486
The GO analyses were performed with method available at the AgriGO website
487
(http://bioinfo.cau.edu.cn) (Du et al., 2010), with the following parameters: Hypergeometric
488
statistical test method, Yekutieli (FDR under dependency) multi-test adjustment method and a
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
15
489
0.05 significance level. Heatmap was obtained using heatmap.2 function of gplots R package
490
(https://cran.r-project.org/web/packages/gplots/index.html) using Pearson distance and an
491
average method for the hierarchical clustering.
492
493
qPCR validation
494
For qPCR validation, 500 ng of total or polysomal RNA was reverse-transcribed with
495
SuperScript IV Kit (Life TechnologyTM) using Oligo dT18. Real-time PCR was performed in
496
an LC 480 384-well thermocycler. The following program was used: 5 min 95°C, 40 cycles of
497
15 s at 95°C, and 1 min at 60°C). The PCR mix contained TakyonTM qPCR master mix
498
(Eurogentec), 500 nM gene-specific primers, and 1.8 μL cDNA in a total volume of 9 μL.
499
Primer efficiencies were determined on standard curves. Primers used in this study are present
500
in Supplemental Table S5.
501
502
RNA co-Immunoprecipitation
503
RNA co-Immunoprecipitation was performed as described previously using the UBP1a-GFP
504
line or Col0 line as a negative control (Sorenson and Bailey-Serres, 2014). Briefly, 400 mg of
505
tissue powder were incubated in 3 mL of lysis buffer (200 mM Tris pH 9.0, 110 mM
506
Potassium Acetate, 0.5 % triton-X100, 0.1% Tween-20, 5 mM DTT, 1.5% Protease
507
Inhibitor). Lysate was clarified using Miracloth (MilliporeTM) layer and centrifuged at 1,500 g
508
during 2 min at 4°C. 1.5 mL of crude extract was incubated with 15 μL of GFP-trap® beads
509
(ChromoTek) during 1,5 h at 4°C under rotation. 0.75 mL of crude extract was incubated with
510
7.5 μL of GFP-trap beads for western blot analysis. Beads were washed five times with 0.75
511
mL of lysis buffer. For RIP experiments, elution was performed with 200 μL of 8M
512
guanidium for 5 min on ice and precipitated overnight with 300 μL of 100% ethanol before
513
RNA extraction as described above. For western blot, elution was performed by adding 20 μL
514
of Laemmli 2X to beads and incubated 5 min at 95°C. Reverse transcription was performed
515
on 10 μL of eluate or 500 ng of input as described above. 1 μL of cDNA was used for PCR
516
amplification with specific primers (Supplemental Table S5).
517
518
15
519
After heat treatment (as described above), seedlings were transferred in a liquid medium
520
containing 1mM KH2PO4, 50 μM FeNaEDTA, 0.5 mM MgCl2, 0.5 mM CaCl2 2H2O, 10 mM
521
KNO3. For
522
performed during 2 h recovery. For transcription inhibition, actinomycin D was added to the
N Incorporation and Elemental Analysis
15
N incorporation, KNO3 was replaced by 99% K15NO3. Incorporation was
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
16
523
medium at 0.2 mg.mL-1 during recovery. After incorporation, polysomes were extracted as
524
described previously. Polysomal proteins were precipitated 6 h at 4°C by adding two volumes
525
of 100% ethanol. After centrifugation, pellets were dried at 70°C for 48 hours and analyzed
526
for total N content and atom % 15N abundance by Continuous-Flow Mass Spectrometry, using
527
a Euro-EA Elemental Analyzer (Eurovector SPA, Redavalle, Italy) coupled with an IsoPrime
528
mass spectrometer (Isoprime Ltd,Stockport, UK).
529
530
Accession number
531
The accession number for the RNAseq data reported in this paper is NCBI Bioproject
532
PRJNA345276.
533
534
Acknowledgments
535
We thank the Academia Sinica NGS Core Facility for libraries preparation and RNA
536
sequencing. RM was the recipient of an Academia Sinica Postdoctoral Research Fellowship.
537
We thank Dr. Frédéric Pontvianne and Pr. Julia Bailey-Serres for kindly giving us FIB2-YFP
538
and UBP1a-GFP lines respectively. This work was supported by CNRS and UVPD through
539
utilization of confocal microscope and qPCR devices at the Technoviv platform.
540
541
Figure legends
542
543
Figure 1. hsp101 knockout mutant is affected in polysome recovery. A-F. Polysome
544
profiles on 7 day-old seedlings in wild type (A, B, C) or hsp101 (D, E, F). A, D. Control
545
22°C. B, E. heat shock (HS) for 1 h at 37°C. C, F. 2 h of recovery at 22°C after HS. G.
546
Percentage of polysomes for each condition. N=3 biological repeats, mean ± SD. A t-test was
547
performed between wild type and hsp101 for control (p>0.1), at 1 h 37°C (p>0.1) and at 2 h
548
recovery (p<0.05).
549
550
Figure 2. hsp101 knockout mutant is affected in stress granules dissociation after HS. A-
551
L. Monitoring of SG formation using pPABP2-RFP-PABP2 in wild type (A-F) or the hsp101
552
background (G-L). A, D, G, J. Control at 22°C. B, E, H, K. HS for 1 h at 37°C. C, F, I, L. 2
553
h recovery at 22°C after HS. Pictures are representative of at least three independent analyses.
554
White lines correspond to 10 μm. M. SG quantification in wild type and hsp101 after 1 h
555
37°C and after 2 h recovery. N=5 biological repeats, mean ± SD. A t-test was performed
556
between wild type and hsp101 at 1 h 37°C (p>0.1), at 2 h recovery (p<0.001) and for hsp101
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
17
557
between 1 h 37°C and 2 h recovery (p<0.01). Quantification was performed on the same
558
volume of stacks for each condition.
559
560
Figure 3. Translation efficiency of mRNAs released from translation during HS is
561
affected in the hsp101 mutant during recovery phase. A. Workflow used to identify
562
transcripts stored in SG and released from translation during recovery. Only mRNAs with an
563
FC between 0.5 and 2 on total RNA population and FC below 0.5 on polysomal RNA
564
population in wt and hsp101 were kept. B, C. TE of the 3309 mRNAs during HS vs. recovery
565
period in wt (B) and hsp101 (C). D. The log2 value of TE of the 3309 mRNAs during
566
recovery period in wt vs. hsp101. Red dots correspond to mRNAs affected in translation
567
recovery in hsp101 (2103). The black line corresponds to a linear regression of the 3309 dots.
568
569
Figure 4. Translation efficiency of cytoplasmic RP mRNAs in wild type and hsp101
570
under normal condition, after 1 h at 37°C and after 2 h recovery. Heatmap of cytoplasmic
571
RP mRNAs translation efficiency in log2 value. The Heatmap was performed on the 219
572
cytoplasmic RP mRNAs identified in RNAseq database. 179 of them are grouped in the same
573
cluster. The numbers of transcripts and the mean value of TE of this cluster are marked. Red
574
values correspond to a high translation efficiency and green values to a low translation
575
efficiency.
576
577
Figure 5. Determination of RP mRNAs translation efficiency by qPCR. A, B. Translation
578
efficiency of the At5g02960 and At3g44010 genes determined by qPCR. Translation
579
efficiency was determined as the ratio of mRNAs quantity between polysomal and total RNA
580
values. Values are normalized to ACTIN7 level. N=3 biological repeats, Mean ± SD.
581
582
Figure 6. RP mRNAs are associated with UBP1 during HS and released after stress. A.
583
Western blot analysis of UBP1a-GFP immunoprecipitation efficiency using GFP antibody. B,
584
C, D, E. PCR amplification (30 cycles) of the At3g44010 (RPS29B), At5g02960 (RPS23B),
585
At1g52300 (RPL37B) and At5g09810 (ACTIN7) genes after RNA immunoprecipitation with
586
the 35S-UBP1a-GFP line. Wild type line was used as a negative control. 1. Input UBP1a. 2.
587
Input wild type. 3. Eluate UBP1a. 4. Eluate wild type.
588
589
Figure 7. New ribosomes can be produced during recovery independently of 40S and
590
60S reassembly. A. Polysome profiles analysis in wild type after HS 1 h at 37°C, and after 30
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
18
591
min, 1 h and 2 h recovery at 22°C. Polysome profiles analysis was performed three times on
592
three biological repeats. The area of 40/60S and 80S/polysomes were determined for each
593
condition and normalized according to the respective area for HS condition. Data are
594
presented below each profile. N=3 biological repeats, Mean ± SD. B. Polysome recovery after
595
HS on liquid medium supplemented with actinomycin D. C. 15N incorporation in polysomal
596
proteins after 2 h recovery with or without actinomycin D treatment, N=3 biological repeats,
597
mean ± SD. Puromycin treatment was used as a negative control. Values were normalized by
598
15
N natural abundance.
599
600
Supplemental data
601
602
Figure S1. Polysome profiles after 2 h of recovery in wild type (A), dlt1-1 (B), hsp101 (C)
603
and dlt1-2 (D). Data correspond to the same biological repeats. dlt1-1 is a missense mutant of
604
HSP101 with little effect on HSP101 chaperone activity whereas dlt1-2 is a missense of
605
HSP101 that impairs HSP101 chaperone activity.
606
607
Figure S2. A. Western blot analysis of RFP-PABP2 lines. Levels of HSP101, PABP, and
608
ACTIN were checked using specific antibodies. αPABP recognizes PABP2, PABP4, and
609
PABP8 proteins. 1. wild type. 2. hsp101. 3. pPABP2-RFP-PABP2 in wild type. 4. pPABP2-
610
RFP-PABP2 in hsp101 background. B. Monitoring of SG formation using pPABP2-RFP-
611
PABP2 in wild type or in hsp101 background after 2 h recovery. 5 different roots are
612
represented. White lines correspond to 10 μm.
613
614
Figure S3. Log2 value of the 3309 mRNAs in control (A) and after 1 h at 37°C (B) in wild
615
type vs. hsp101. Black line correspond to the linear regression of the gray dots.
616
617
Figure S4. Gene Ontology (GO) analysis of mRNAs poorly translated during HS and affected
618
in translation recovery in hsp101 (2103/3309). GO groups highly enriched are presented in
619
red. p-values are present in parentheses for all groups.
620
621
Figure S5. Translation efficiency of chloroplastic and mitochondrial RP mRNAs in wild type
622
and hsp101 under normal condition, after 1 h at 37°C and after 2 h recovery. A. Heatmap of
623
chloroplastic RP mRNAs translation efficiency in log2 value. The Heatmap was performed on
624
the 50 chloroplastic RP mRNAs identified in RNAseq database. 27 of them are grouped in the
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
19
625
same cluster. B. Heatmap of mitochondrial RP mRNAs translation efficiency in log2 value.
626
The numbers of transcripts and the mean value of TE are marked. Red values correspond to a
627
high translation efficiency and green values to a low translation efficiency.
628
629
Figure S6. Heat stress did not affect nucleoli architecture. A, B, C. Confocal microscopy
630
analysis of nucleoli architecture using FIB2-YFP in normal condition (A), after 1 h at 37°C
631
(B), and after 1 h at 22°C in liquid medium supplemented with actinomycin D (C). White
632
lines correspond to 10 μm. Pictures are representative of 25 nucleoli analyzed on 5 different
633
seedlings (5 per seedlings) for each condition.
634
635
636
Figure S1. Polysome profiles after 2 h of recovery in wild type (A), dlt1-1 (B), hsp101 (C)
637
and dlt1-2 (D).
638
Figure S2. A. Western blot analysis of RFP-PABP2 lines.
639
Figure S3. Log2 value of the 3309 mRNAs in control (A) and after 1 h at 37°C (B) in wild
640
type vs. hsp101.
641
Figure S4. Gene Ontology (GO) analysis of mRNAs poorly translated during HS and affected
642
in translation recovery in hsp101 (2103/3309).
643
Figure S5. Translation efficiency of chloroplastic and mitochondrial RP mRNAs in wild type
644
and hsp101 under normal condition, after 1 h at 37°C and after 2 h recovery.
645
Figure S6. Heat stress did not affect nucleoli architecture. A, B, C.
646
647
Table S1. RNA sequencing raw data
648
649
Table S2. Transcripts stable and released from translation during HS in wt and hsp101
650
(3309)
651
652
Table S3. Transcripts affected in translation recovery in hsp101 (2103)
653
654
Table S4. Translation Efficiency of Ribosomal Proteins mRNAs
655
656
Table S5. Primers used in this study
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
20
657
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Merret R, Nagarajan VK, Carpentier MC, Park S, Favory JJ, Descombin J, Picart C,
721
Charng YY, Green PJ, Deragon JM, et al (2015) Heat-induced ribosome pausing
722
triggers mRNA co-translational decay in Arabidopsis thaliana. Nucleic Acids Res 43:
723
4121–4132
724
Nicolaï M, Roncato M a, Canoy a S, Rouquié D, Sarda X, Freyssinet G, Robaglia C
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
22
725
(2006) Large-scale analysis of mRNA translation states during sucrose starvation in
726
arabidopsis cells identifies cell proliferation and chromatin structure as targets of
727
translational control. Plant Physiol 141: 663–673
728
Nover L, Mumsche D, Neumann D, Ohme K, Scharf K (1986) Control of ribosome
729
biosynthesis in plant cell cultures under heat-shock conditions. Eur J Biochem 160: 297–
730
304
731
Park S-H, Chung PJ, Juntawong P, Bailey-Serres J, Kim YS, Jung H, Bang SW, Kim Y-
732
K, Do Choi Y, Kim J-K (2012) Posttranscriptional Control of Photosynthetic mRNA
733
Decay under Stress Conditions Requires 3’ and 5’ Untranslated Regions and Correlates
734
with Differential Polysome Association in Rice. Plant Physiol 159: 1111–1124
735
Perea-Resa C, Carrasco-López C, Catalá R, Turečková V, Novak O, Zhang W, Sieburth
736
L, Jiménez-Gómez JM, Salinas J (2016) The LSM1-7 Complex Differentially
737
Regulates Arabidopsis Tolerance to Abiotic Stress Conditions by Promoting Selective
738
mRNA Decapping. Plant Cell 28: 505–520
739
740
Picart C, Pontvianne F (2016) Plant nucleolar DNA: green light shed on the role of
Nucleolin in genome organization. Nucleus 0
741
Pontvianne F, Blevins T, Chandrasekhara C, Mozgová I, Hassel C, Pontes OMF, Tucker
742
S, Mokroš P, Muchová V, Fajkus J, et al (2013) Subnuclear partitioning of rRNA
743
genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes
744
Dev 27: 1545–1550
745
746
Protter DSW, Parker R (2016) Principles and Properties of Stress Granules. Trends Cell
Biol 26: 668–679
747
Puckette M, Iyer NJ, Tang Y, Dai X Bin, Zhao P, Mahalingam R (2012) Differential
748
mRNA translation in medicago truncatula accessions with contrasting responses to
749
ozone-induced oxidative stress. Mol Plant 5: 187–204
750
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch
751
S, Rueden C, Saalfeld S, Schmid B, et al (2012) Fiji: an open-source platform for
752
biological-image analysis. Nat Methods 9: 676–682
753
Sorenson R, Bailey-Serres J (2014) Selective mRNA sequestration by
754
OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during
755
hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 111: 2373–8
756
Ueda K, Matsuura H, Yamaguchi M, Demura T, Kato K (2012) Genome-wide analyses of
757
changes in translation state caused by elevated temperature in Oryza sativa. Plant Cell
758
Physiol 53: 1481–1491
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
23
759
Wallace EWJ, Kear-Scott JL, Pilipenko E V., Schwartz MH, Laskowski PR, Rojek AE,
760
Katanski CD, Riback JA, Dion MF, Franks AM, et al (2015) Reversible, Specific,
761
Active Aggregates of Endogenous Proteins Assemble upon Heat Stress. Cell 162: 1286–
762
1298
763
764
765
766
767
Walters RW, Parker R (2015) Coupling of Ribostasis and Proteostasis: Hsp70 Proteins in
mRNA Metabolism. Trends Biochem Sci 40: 552–559
Weber C, Nover L, Fauth M (2008) Plant stress granules and mRNA processing bodies are
distinct from heat stress granules. Plant J 56: 517–530
Wu T-Y, Juan Y-T, Hsu Y-H, Wu S-H, Liao H-T, Fung RWM, Charng Y-Y (2013)
768
Interplay between Heat Shock Proteins HSP101 and HSA32 Prolongs Heat Acclimation
769
Memory Posttranscriptionally in Arabidopsis. Plant Physiol 161: 2075–2084
770
Xu J, Chua N-H (2009) Arabidopsis decapping 5 is required for mRNA decapping, P-body
771
formation, and translational repression during postembryonic development. Plant Cell
772
21: 3270–9
773
Xu J, Yang J-Y, Niu Q-W, Chua N-H (2006) Arabidopsis DCP2, DCP1, and VARICOSE
774
form a decapping complex required for postembryonic development. Plant Cell 18:
775
3386–98
776
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
24
Figure1
Control
OD254nm
wildtype
1h37°C
A
2hrecovery
B
C
E
F
80S
40S
60S
D
OD254nm
hsp101
Polysomes
GG
H
wildtype
hsp101
II
%Polysomes
50
*
25
0
Control
1h37°C
2hrecovery
Figure 1. hsp101 knockout mutant is affected in polysome recovery. A-F. Polysome profiles on 7 day-old
seedlings in wild type (A, B, C) or hsp101 (D, E, F). A, D. Control 22°C. B, E. heat shock (HS) for 1 h at 37°C.
C, F. 2 h of recovery at 22°C after HS. G. Percentage of polysomes for each condition. N=3 biological repeats,
mean ± SD. A t-test was performed between wild type and hsp101 for control (p>0.1), at 1 h 37°C (p>0.1) and at
2 h recovery (p<0.05). Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Control
Figure2
wildtype
2hrecovery
A
B
C
D
E
F
G
H
I
J
K
L
Numberofstressgranules
hsp101
1h37°C
4000
wildtype
H
M
hsp101
**
3000
***
2000
1000
0
1h37°C
2hrecovery
Figure 2. hsp101 knockout mutant is affected in stress granules dissociation after HS. A-L. Monitoring of SG formation
using pPABP2-RFP-PABP2 in wild type (A-F) or the hsp101 background (G-L). A, D, G, J. Control at 22°C. B, E, H, K. HS
for 1 h at 37°C. C, F, I, L. 2 h recovery at 22°C after HS. Pictures are representative of at least three independent analyses.
White lines correspond to 10 µm. M. SG quantification in wild type and hsp101 after 1h 37°C and after 2 h recovery. N=5
biological repeats, mean ± SD. A t-test was performed between wild type and hsp101 at 1 h 37°C (p>0.1), at 2 h recovery
(p<0.001) and for hsp101 between 1 h 37°C and 2 h recovery (p<0.01). Quantification was performed on the same volume of
stacks for each condition. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Figure3
FC37°C/ControlTotal[0.5;2]
896
10662
1117
3
D
2
1
wt
0
0
wt
hsp101
1
2
3
TE1h37°C
4
FC37°C/ControlPolysomes<0.5
327
wt
3309
1188
hsp101
TE2hrecovery
C 4
3
TE2hrecoveryhsp101(log2)
A
TE2hrecovery
B 4
1,5
0
-1,5
3
2
y=0.8041x
-3
-3
1
hsp101
-1,5
0
1,5
3
TE2hrecoverywt(log2)
0
0
1
2
3
TE1h37°C
4
Figure 3. Translation efficiency of mRNAs released from translation during HS is affected in the hsp101 mutant
during recovery phase. A. Workflow used to identify transcripts stored in SG and released from translation during
recovery. Only mRNAs with an FC between 0.5 and 2 on total RNA population and FC below 0.5 on polysomal RNA
population in wt and hsp101 were kept. B, C. TE of the 3309 mRNAs during HS vs. recovery period in wt (B) and hsp101
(C). D. The log2 value of TE of the 3309 mRNAs during recovery period in wt vs. hsp101. Red dots correspond to
mRNAs affected in translation recovery in hsp101 (2103). The black line corresponds to a linear regression of the 3309
dots.
Figure4
Control
1h37°C
2hrecovery
179/219
Log2
-1
0
1
wt
hsp101
wt
hsp101
wt
hsp101
Figure 4. Translation efficiency of cytoplasmic RP mRNAs in wild type and hsp101 under normal condition, after 1 h at
37°C and after 2 h recovery. Heatmap of cytoplasmic RP mRNAs translation efficiency in log2 value. The Heatmap was
performed on the 219 cytoplasmic RP mRNAs identified in RNAseq database. 179 of them are grouped in the same cluster. The
numbers of transcripts and the mean value of TE of this cluster are marked. Red values correspond to a high translation
efficiency and green values to a low translation efficiency. Figure5
A
6
wildtype
4
2
0
Control
1h37°C
2hrecovery
At3g44010:RPS29B
6
hsp101
Transla2onEfficiency
Transla2onEfficiency
B
At5g02960:RPS23B
wildtype
hsp101
4
2
0
Control
1h37°C
2hrecovery
Figure 5. Determination of RP mRNAs translation efficiency by qPCR. A, B. Translation efficiency of the At5g02960
and At3g44010 genes determined by qPCR. Translation efficiency was determined as the ratio of mRNAs quantity between
polysomal and total RNA values. Values are normalized to ACTIN7 level. N=3 biological repeats, Mean ± SD. Figure 6
Control
1 h 37°C
2 h recovery
A
αGFP
B
RPS23B
C
RPS29B
D
RPL37B
E
ACTIN7
1 2 3 4
1 2 3 4
1 2 3 4
Figure 6. RP mRNAs are associated with UBP1 during HS and released after stress. A.
Western blot analysis of UBP1a-GFP immunoprecipitation efficiency using GFP antibody.
B, C, D, E. PCR amplification (30 cycles) of the At5g02960 (RPS23B), At3g44010
(RPS29B), At1g52300 (RPL37B) and At5g09810 (ACTIN7) genes after RNA
immunoprecipitation with the 35S-UBP1a-GFP line. Wild type line was used as a negative
control. 1. Input UBP1a. 2. Input wild type. 3. Eluate UBP1a. 4. Eluate wild type.
Figure7
1h37°C
30’recovery
1hrecovery
2hrecovery
OD254nm
A
1
B
1.08
±
0.03
1.1
±
0.01
1.14
±
0.09
OD254nm
C
E
D
1h37°C
1.65
±
0.2
1hrecoveryActD
2hrecoveryActD
15NAmount(μmoles/gDW)
1
0.94
±
0.06
8
1.6
±
0.2
6
4
2
0
15N
15N
F
15N
ActD+15N
PPuromycin
uromycin
+ActD
Figure 7. New ribosomes can be produced during recovery independently of 40S and 60S reassembly. A.
Polysome profiles analysis in wild type after HS 1 h at 37°C, and after 30 min, 1 h and 2 h recovery at 22°C.
Polysome profiles analysis was performed three times on three biological repeats. The area of 40/60S and 80S/
polysomes were determined for each condition and normalized according to the respective area for HS condition.
Data are presented bellow each profile. N=3 biological repeats, Mean ± SD. B. Polysome recovery after HS on liquid
medium supplemented with actinomycin D. C.
15N
incorporation in polysomal proteins after 2 h recovery with or
without actinomycin D treatment, N=3 biological repeats, mean ± SD. Puromycin treatment was used as a negative
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