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Alu RNA Regulates the Cellular Pool of Active Ribosomes by Targeted Delivery of SRP9/14 to 40S Subunits

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Alu RNA Regulates the Cellular Pool of Active Ribosomes by Targeted Delivery of SRP9/14 to 40S Subunits Article Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits IVANOVA, Elena, et al. Abstract The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition in- volves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases transla- tion of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mR- NAs resuming [...] Reference IVANOVA, Elena, et al. Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits. Nucleic Acids Research, 2015, vol. 43, no. 5, p. 2874-2887 DOI : 10.1093/nar/gkv048 Available at: http://archive-ouverte.unige.ch/unige:55879 Disclaimer: layout of this document may differ from the published version. 1 / 1 Nucleic Acids Research Advance Access published February 19, 2015 Nucleic Acids Research, 2015 1 doi: 10.1093/nar/gkv048 Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits Elena Ivanova1,*,AudreyBerger1,AnneScherrer1, Elena Alkalaeva2 and Katharina Strub1,* 1Departement´ de biologie cellulaire, UniversitedeGen´ eve,` Sciences III, 1211 Geneve,` Switzerland and 2Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia Received October 16, 2014; Revised January 08, 2015; Accepted January 12, 2015 ABSTRACT tional control may occur at different steps during protein synthesis, the majority of the mechanisms target translation The human genome contains about 1.5 million Alu initiation (1,2). Downloaded from elements, which are transcribed into Alu RNAs by The canonical pathway for translation initiation proceeds RNA polymerase III. Their expression is upregulated through sequential formation of several initiation com- following stress and viral infection, and they as- plexes (3). Ternary complex and the initiation factors bind sociate with the SRP9/14 protein dimer in the cy- to the 40S ribosomal subunit yielding the 43S preinitiation toplasm forming Alu RNPs. Using cell-free transla- complex, which is then loaded onto the 5′-proximal region http://nar.oxfordjournals.org/ tion, we have previously shown that Alu RNPs inhibit of an mRNA and scans for the initiation codon. Once the polysome formation. Here, we describe the mecha- initiation codon is positioned at the P-site, the 48S initia- nism of Alu RNP-mediated inhibition of translation tion complex is assembled. The joining of the 60S ribosomal initiation and demonstrate its effect on translation subunit leads to formation of the 80S initiation complex, which then proceeds to elongation cycles. of cellular and viral RNAs. Both cap-dependent and Translation initiation is regulated by the orchestrated ac- IRES-mediated initiation is inhibited. Inhibition in- tion of multiple control mechanisms, many of which re- volves direct binding of SRP9/14 to 40S ribosomal main to be elucidated. We have previously shown that the subunits and requires Alu RNA as an assembly fac- noncoding, retrotransposon-derived Alu RNA in complex by guest on February 27, 2015 tor but its continuous association with 40S subunits with the heterodimeric protein SRP9/14 (forming together is not required for inhibition. Binding of SRP9/14 to the Alu RNP) is able to diminish polysome formation in a 40S prevents 48S complex formation by interfering wheat germ translation system and thus, most likely inter- with the recruitment of mRNA to 40S subunits. In feres with translation initiation (4). These data raised the cells, overexpression of Alu RNA decreases transla- intriguing possibility that Alu RNPs are involved in a novel tion of reporter mRNAs and this effect is alleviated translational control pathway. with a mutation that reduces its affinity for SRP9/14. Alu elements are derived from the 7SL RNA gene (5), which encodes the RNA moiety of the signal recognition Alu RNPs also inhibit the translation of cellular mR- particle (SRP), and following retrotransposition Alu ele- NAs resuming translation after stress and of viral ments now make up over 10% of the human genome mass. mRNAs suggesting a role of Alu RNPs in adapting The modern Alu element is composed of two similar, but the translational output in response to stress and vi- not identical monomers named the left arm and the right ral infection. arm (Figure 1A). Based on mutations at diagnostic posi- tions, Alu elements are divided into three families of dif- ferent evolutionary ages (8). The most ancient family is INTRODUCTION named AluJ followed by AluS and AluY. Alu elements can Translation regulation ensures protein homeostasis under be part of a polymerase II transcription unit and are there- normal growth conditions. In addition, modulating the fore present in pre-mRNAs and mRNAs. These embedded translation effciency of already existing mRNAs provides Alu sequences can infuence gene expression in many ways a rapid mechanism for adapting protein expression in de- for example by providing alternative splice sites as well as velopment, memory formation, synaptic plasticity and in target sites of miRNAs and editing enzymes (6). response to stress and apoptotic signals. Although transla- *To whom correspondence should be addressed. Tel : +41 22 379 67 24; Email: [email protected] Correspondence may also be addressed to Elena Ivanova. Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. ⃝ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Nucleic Acids Research, 2015 A B Alu RNP A AC AluY RNP C U G U ]%[ A A U 40 CG scAluY RNP CG 50 GG 70 CG UC UA C A G A A G A A G A G G CC GAGGCGGG UCAC GGUCAGGA A SRP9/14 80 noi .ffe AluY RNA U C C GG CUCUGCCC CGGUCCU G G AGUG U A A Left arm U G UA C A G A C C UG C 110 A U A 90 A CG 5' U 100 AC A scAluY RNA GC A 120 20 C G A AC 10 A CG A scAlu RNP U G T C A h9/14 U C GG * A A talsnar A A 130 C U AC A A 170 G U A A C A CG U 230 C G U 190 200 220 C G 180 A 210 GG U A U A AA GAA A C CU A G GGC GAGGC GGAG UGGCGU CCCUGG GGAG UGCAGUG GCCGA SRP9/14 A G U CCG A t G CUCUG CC UC AGCGAGA CGGGUCC CCUC AGUCAC CCGGUU Right arm U G A21 AG C G GA C CG140 280 270 A 260 250 CG 3' 240 GC C G GU GG G A U U GG * 150 [nM] C D E RRL + edeine 0.5 µM 48S assembly 5’ E P A 3’ 120 10000 AMV Buffer 100 5000 5’ E P A 3’ 0 80 AMV 10000 60 AluY A RNP 16-18 nt 5000 40 0 10000 translation eff. [%] 20 scAluY A RNP Downloaded from 0 Fragment analysis 5000 A A ABI PRISM 0 Buffer AluY 10000 scAluY scAluY A RNA 100 nM 5000 Relative fluorescence units 0 A F AluY RNP 10000 h9/14 A 120 scAluY RNP 5000 A http://nar.oxfordjournals.org/ 100 scAluY RNA 0 80 h9/14 5’-end unspecific peaks 48S 60 40 H 150 Buffer 20 AluYA 48S assembly eff. [%] 48S assembly eff. 0 scAluYA 0 200 400 600 800 1000 100 [nM] G 50 by guest on February 27, 2015 80S assembly Elongation 500 3000 Buffer Buffer [%] 48S assembly eff. 2000 300 0 1000 100 0 48S 80S pre TC 0 500 3000 AluYA RNP AluYA RNP 300 I 120 2000 1000 100 0 100 0 500 3000 scAluYA RNP scAluYA RNP 80 2000 300 Relative fluorescence units 1000 100 60 Relative fluorescence units 0 0 40 unspecific peaks unspecific peaks 80S 80S 5’-end 5’-end preTC 20 48S assembly eff. [%] 48S assembly eff. 0 43S 48S + Alu RNP Figure 1. Alu RNPs repress translation initiation by inhibiting 48S complex formation. (A)SecondarystructuremodelofAluYA RNA. Binding sites of h9/14 are indicated by analogy to SRP RNA. Preparation of Alu RNPs is described in Supplementary Figure S1. (B)Translationeffciencies of uncapped pPL mRNA in RRL supplemented with increasing concentrations of Alu RNPs or the h9/14 protein or the AluYA/scAluYA RNAs alone. [35S]-labeled proteins were resolved by SDS-PAGE and quantifed by phosphorimager (see Supplementary Figure S2A and B for SDS-PAGE autoradiographs). Trans- lation effciency was calculated as a percentage of the value obtained in the absence of RNPs. Error bars are shown as SD (n 3). (C)Translationeffciencies of uncapped pPL mRNA in RRL translation reaction synchronized by 0.5 ␮M edeine in the presence of 100 nM AluYA/scAluY≥ A RNP.
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