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Eif4g2 Balances Its Own Mrna Translation Via a PCBP2-Based Feedback Loop

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Eif4g2 Balances Its Own Mrna Translation Via a PCBP2-Based Feedback Loop Downloaded from rnajournal.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT eIF4G2 balances its own mRNA translation via a PCBP2-based feedback loop VICTORIA V. SMIRNOVA,1,2 EKATERINA D. SHESTAKOVA,3 DMITRY V. BIKMETOV,1 ANASTASIA A. CHUGUNOVA,4,5 ILYA A. OSTERMAN,2,4,5 MARINA V. SEREBRYAKOVA,2 OLGA V. SERGEEVA,5 TIMOFEY S. ZATSEPIN,5 IVAN N. SHATSKY,2 and ILYA M. TERENIN2,6 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskie Gory, 119234 Moscow, Russia 2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119992, Russia 3Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow, 119234, Russian Federation 4Chemistry Department, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russia 5Skolkovo Institute of Science and Technology, Skolkovo, Moscow Region 143026, Russia 6Sechenov First Moscow State Medical University, Institute of Molecular Medicine, 119991, Moscow, Russian Federation ABSTRACT Poly(rC)-binding protein 2 (PCBP2, hnRNP E2) is one of the most abundant RNA-binding proteins in mammalian cells. In humans, it exists in seven isoforms, which are assumed to play similar roles in cells. The protein is shown to bind 3′′′′′-untrans- lated regions (3′′′′′-UTRs) of many mRNAs and regulate their translation and/or stability, but nothing is known about the func- tional consequences of PCBP2 binding to 5′′′′′-UTRs. Here we show that the PCBP2 isoform f interacts with the 5′′′′′-UTRs of mRNAs encoding eIF4G2 (a translation initiation factor with a yet unknown mechanism of action, also known as DAP5) and Cyclin I, and inhibits their translation in vitro and in cultured cells, while the PCBP2 isoform e only affects Cyclin I trans- lation. Furthermore, eIF4G2 participates in a cap-dependent translation of the PCBP2 mRNA. Thus, PCBP2 and eIF4G2 seem to regulate one another’s expression via a novel type of feedback loop formed by the translation initiation factor and the RNA-binding protein. Keywords: translational control; ribosomal scanning; αCP2 INTRODUCTION Hundsdoerfer et al. 2005; Lewis et al. 2008; Marash et al. 2008), although a stimulatory action of eIF4G2 on cap-de- The mechanism of translation initiation in eukaryotes pendent translation was also reported (Lee and McCormick was established about two decades ago when Marilyn 2006; de la Parra et al. 2018). The protein is essential for de- Kozak’s scanning model was complemented with a bio- velopment in mouse (Yamanaka et al. 2000), zebrafish chemical characterization of the initiation factors’ activities (Nousch et al. 2007), and Drosophila (Yoshikane et al. (for reviews, see Hinnebusch 2011, 2014). Since then, this 2007), probably because of its participation in the transla- basement has not been shaken, although there are numer- tion of differentiation-associated proteins (Yoffe et al. ous cases that do not fit this model particularly well. eIF4G2 2016; Sugiyama et al. 2017), and its overexpression in ES (also DAP5, Nat1) is one of such square pegs in a round hole cells leads to a spontaneous differentiation (Takahashi of our knowledge of eukaryotic translation initiation. The et al. 2005). Remarkably, to date, no organism is known protein seems to be present in all Chordata and many whose eIF4G2 mRNA uses an AUG triplet as an initiator co- (but clearly not all) invertebrates. Despite its close homolo- don. In apparently most (if not all) vertebrates, it is GUG, gy with eIF4G1, eIF4G2 lacks binding sites for eIF4E and while in Insecta, Mollusca, or simple Chordata, no transla- PABP (Imataka et al. 1997; Levy-Strumpf et al. 1997; tion initiation site can be assigned unambiguously. Such Shaughnessy et al. 1997; Yamanaka et al. 1997) and thus an evolutionarily conserved mRNA feature implies the it is believed to have a hand in cap-independent translation of certain cellular mRNAs (Henis-Korenblit et al. 2002; © 2019 Smirnova et al. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publica- Corresponding authors: [email protected], shatsky@ tion date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After belozersky.msu.ru 12 months, it is available under a Creative Commons License Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. (Attribution-NonCommercial 4.0 International), as described at http:// 065623.118. creativecommons.org/licenses/by-nc/4.0/. RNA (2019) 25:757–767; Published by Cold Spring Harbor Laboratory Press for the RNA Society 757 Downloaded from rnajournal.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Smirnova et al. existence of posttranscriptional regulation. It is now widely for the regulation of eIF4G2 expression, we included it recognized that any mRNA is heavily packed with the and started 30 nucleotides (nt) of the eIF4G2 coding re- bound proteins (Baltz et al. 2012; Castello et al. 2012; gion in the reporter. Additionally, the firefly luciferase Beckmann et al. 2015; Hentze et al. 2018), and since every AUG codon was mutated to exclude any contribution of known case of posttranscriptional regulation relies on the translation initiation downstream from the suboptimal mRNA-binding proteins, we sought to identify the proteins authentic eIF4G2 start codon. that interact with the human eIF4G2 mRNA. In vitro transcribed noncapped biotinylated RNA, corre- Poly(rC)-binding proteins (hnRNP K and PCBP1-4) are sponding to the human eIF4G2 5′-UTR, was used to pull important actors in mRNA metabolism. They participate down proteins from HeLa cytoplasmic extract (Fig. 1, left in splicing (Expert-Bezançon et al. 2002; Ji et al. 2016; lane). The proteins specifically bound to the eIF4G2 Grelet et al. 2017), transcription (Choi et al. 2009), transla- 5′-UTR were identified by mass spectrometry as hnRNP tional repression, and stabilization or destabilization of K, HuR, SERBP1, YBX3, PCBP2, and PTBP1. YBX1 and mRNAs via an interaction with their 3′-UTRs (Scoumanne hnRNP Q are ubiquitously bound to any RNA tested (Fig. et al. 2011; Han et al. 2013; Ren et al. 2016). The two prob- 1; also, data not shown). Similarly to the case of poliovirus ably most known cases of their translation-related activities IRES (Blyn et al. 1995, 1996, 1997; Andreev et al. 2012), are the stabilization of α-globin mRNA and the repression PCBP2 manifests itself in the multiple bands, reflecting of ALOX15 mRNA translation during hematopoiesis (for the diversity of its isoforms. review, see Ostareck-Lederer and Ostareck 2012). PCBP2 also participates in translation driven by the IRES element PCBP2f inhibits translation of the reporter mRNA from poliovirus and other related viruses (Blyn et al. 1997; bearing the eIF4G2 5′′′′′-UTR in vitro Gamarnik and Andino 1997; Graff et al. 1998; Walter et al. 1999; Sweeney et al. 2014; Asnani et al. 2016a). However, All the identified proteins except for YBX3, the cDNA ′ cases in which PCBP2 affects 5 -end-dependent transla- of which we have failed to amplify from a dozen differ- ′ tion via binding to 5 -UTRs, are not explicitly described. ent cDNA preparations, were cloned and expressed in In humans, at least seven PCBP2 isoforms (a–g) are known Escherichia coli. We accidentally amplified cDNAs corre- to exist. These isoforms arise from alternative splicing, but sponding to two isoforms of the PCBP2, viz., PCBP2e and all PCBP2-coding mRNAs seem to possess the same PCBP2f. Thus, both were studied. We addressed whether ′ 5 -UTR. To date, no functional difference between the the addition of either protein affects translation of the isoforms has been registered. eIF4G2 reporter mRNA in vitro in Krebs-2 cytoplasmic ′ Here we show that the PCBP2 isoform f binds the 5 -UTR extract. While SERBP1, hnRNP K, HuR, PCBP2e, or PTBP1 of the eIF4G2, and the PCBP2 isoforms e and f bind the exert no specific effect (data not shown) on the translation ′ 5 -UTR of the Cyclin I (CCNI) mRNAs in vitro and inhibit of a dozen of reporter mRNAs tested, including the eIF4G2 the translation of the corresponding reporter mRNAs, while reporter, the other PCBP2 isoform, PCBP2f, inhibits the the PCBP2 knockdown in 293T or Huh7 cells augments their translation. Strikingly, knockdown of the eIF4G2 gene in NIH/3T3, 293T, or Huh7 cells leads to a significantly decreased translation driven by the 5′-UTR of the PCBP2 mRNA as well as the PCBP2 protein level. Thus, eIF4G2 and PCBP2 mutually tune one another’s translation. RESULTS AND DISCUSSION Proteins that bind the 5′′′′′-UTR of the eIF4G2 mRNA First, we sought to identify proteins that interact with the ′ eIF4G2 mRNA. The 5′-UTR and 3′-UTR of the human FIGURE 1. Identification of the proteins bound to the 5 -UTR of the eIF4G2 mRNA (transcript variant 1) were cloned upstream eIF4G2 mRNA. In vitro transcribed biotinylated noncapped RNAs cor- responding to either the human eIF4G2 5′-UTR or its deletion mutant and downstream from the firefly luciferase coding se- lacking the polypyrimidine tract (199–274 nt of the eIF4G2 5′-UTR, quence, respectively, to create a pGL3-eIF4G2 plasmid eIF4G2ΔCU) were soaked in HeLa cytoplasmic extract, the complexes that was used further for the reporter mRNA and the bio- were purified via streptavidin-agarose chromatography, the bound tinylated bait synthesis. The authentic 5′ boundary of the proteins were resolved by SDS-PAGE, and identities of the specific 5′-UTR was defined on the basis of expressed sequence bands were identified by LS-MS. Two major bands that bind to all mRNAs in our hands have been identified as YBX1 and hnRNP tags (EST) and cap-assisted gene expression analysis Q. PCBP2, PTBP1, YBX3, hnRNP K, and SERBP1 were found to inter- (CAGE) data (Severin et al. 2014). Since the evolutionarily act with the polypyrimidine tract within the 5′-UTR of the eIF4G2 conserved GUG initiator codon is most likely important mRNA.
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