eIF3 interacts with histone H4 messenger RNA to regulate its translation
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100578
ISSN1083-351X
AutoresHassan Hayek, Lauriane Gross, Aurélie Janvier, Laure Schaeffer, Franck Martin, Gilbert Eriani, Christine Allmang,
Tópico(s)RNA regulation and disease
ResumoIn eukaryotes, various alternative translation initiation mechanisms have been unveiled for the translation of specific mRNAs. Some do not conform to the conventional scanning-initiation model. Translation initiation of histone H4 mRNA combines both canonical (cap-dependent) and viral initiation strategies (no-scanning, internal recruitment of initiation factors). Specific H4 mRNA structures tether the translation machinery directly onto the initiation codon and allow massive production of histone H4 during the S phase of the cell cycle. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), was shown to selectively recruit and control the expression of several cellular mRNAs. Whether eIF3 mediates H4 mRNA translation remains to be elucidated. Here, we report that eIF3 binds to a stem-loop structure (eIF3-BS) located in the coding region of H4 mRNA. Combining cross-linking and ribonucleoprotein immunoprecipitation experiments in vivo and in vitro, we also found that eIF3 binds to H1, H2A, H2B, and H3 histone mRNAs. We identified direct contacts between eIF3c, d, e, g subunits, and histone mRNAs but observed distinct interaction patterns to each histone mRNA. Our results show that eIF3 depletion in vivo reduces histone mRNA binding and modulates histone neosynthesis, suggesting that synthesis of histones is sensitive to the levels of eIF3. Thus, we provide evidence that eIF3 acts as a regulator of histone translation. In eukaryotes, various alternative translation initiation mechanisms have been unveiled for the translation of specific mRNAs. Some do not conform to the conventional scanning-initiation model. Translation initiation of histone H4 mRNA combines both canonical (cap-dependent) and viral initiation strategies (no-scanning, internal recruitment of initiation factors). Specific H4 mRNA structures tether the translation machinery directly onto the initiation codon and allow massive production of histone H4 during the S phase of the cell cycle. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), was shown to selectively recruit and control the expression of several cellular mRNAs. Whether eIF3 mediates H4 mRNA translation remains to be elucidated. Here, we report that eIF3 binds to a stem-loop structure (eIF3-BS) located in the coding region of H4 mRNA. Combining cross-linking and ribonucleoprotein immunoprecipitation experiments in vivo and in vitro, we also found that eIF3 binds to H1, H2A, H2B, and H3 histone mRNAs. We identified direct contacts between eIF3c, d, e, g subunits, and histone mRNAs but observed distinct interaction patterns to each histone mRNA. Our results show that eIF3 depletion in vivo reduces histone mRNA binding and modulates histone neosynthesis, suggesting that synthesis of histones is sensitive to the levels of eIF3. Thus, we provide evidence that eIF3 acts as a regulator of histone translation. In eukaryotes, translation initiation requires multiple complexes of eukaryotic initiation factors (eIFs) to assemble elongation-competent ribosomes to the mRNA (1Jackson R.J. Hellen C.U.T. Pestova T.V. The mechanism of eukaryotic translation initiation and principles of its regulation.Nat. Rev. Mol. Cell Biol. 2010; 11: 113-127Crossref PubMed Scopus (1515) Google Scholar, 2Valášek L.S. 'Ribozoomin' – translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors (eIFs).Curr. Protein Pept. Sci. 2012; 13: 305-330Crossref PubMed Scopus (86) Google Scholar). The recognition of the m7G cap structure by the eIF4E-binding factor that is part of the translation initiation complex eIF4F (composed of the three subunits eIF4E, eIF4A, and eIF4G) constitutes the first step of the canonical translation initiation and is a prerequisite to ribosomal attachment (3Fortes P. Inada T. Preiss T. Hentze M.W. Mattaj I.W. Sachs A.B. The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation.Mol. Cell. 2000; 6: 191-196Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 4McKendrick L. Thompson E. Ferreira J. Morley S.J. Lewis J.D. Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m(7) guanosine cap.Mol. Cell. Biol. 2001; 21: 3632-3641Crossref PubMed Scopus (95) Google Scholar, 5Sonenberg N. eIF4E, the mRNA cap-binding protein: From basic discovery to translational research.Biochem. Cell Biol. 2008; 86: 178-183Crossref PubMed Scopus (149) Google Scholar). The initiation codon is then recognized by a scanning mechanism of the mRNA by the initiator tRNAiMet linked to the 40S subunit (43S complex). eIF3, the largest multisubunit initiation factor, has been implicated in events throughout the initiation pathway (6Hinnebusch A.G. eIF3: A versatile scaffold for translation initiation complexes.Trends Biochem. Sci. 2006; 31: 553-562Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 7Hinnebusch A.G. Structural insights into the mechanism of scanning and start codon recognition in eukaryotic translation initiation.Trends Biochem. Sci. 2017; 42: 589-611Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 8Aitken C.E. Beznosková P. Vlčkova V. Chiu W.-L. Zhou F. Valášek L.S. Hinnebusch A.G. Lorsch J.R. Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex.Elife. 2016; 5e20934Crossref PubMed Scopus (27) Google Scholar, 9Pestova T.V. Kolupaeva V.G. The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection.Genes Dev. 2002; 16: 2906-2922Crossref PubMed Scopus (375) Google Scholar, 10Karásková M. Gunišová S. Herrmannová A. Wagner S. Munzarová V. Valášek L.S. Functional characterization of the role of the N-terminal domain of the c/Nip1 subunit of eukaryotic initiation factor 3 (eIF3) in AUG recognition.J. Biol. Chem. 2012; 287: 28420-28434Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Bound to the 40S subunit near both the mRNA entry and exit channels, it participates to the stabilization of the 43S preinitiation complex (PIC), to its recruitment to the mRNA (8Aitken C.E. Beznosková P. Vlčkova V. Chiu W.-L. Zhou F. Valášek L.S. Hinnebusch A.G. Lorsch J.R. Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex.Elife. 2016; 5e20934Crossref PubMed Scopus (27) Google Scholar, 11Valášek L.S. Zeman J. Wagner S. Beznosková P. Pavlíková Z. Mohammad M.P. Hronová V. Herrmannová A. Hashem Y. Gunišová S. Embraced by eIF3: Structural and functional insights into the roles of eIF3 across the translation cycle.Nucleic Acids Res. 2017; 45: 10948-10968Crossref PubMed Scopus (50) Google Scholar, 12Obayashi E. Luna R.E. Nagata T. Martin-Marcos P. Hiraishi H. Singh C.R. Erzberger J.P. Zhang F. Arthanari H. Morris J. Pellarin R. Moore C. Harmon I. Papadopoulos E. Yoshida H. et al.Molecular landscape of the ribosome pre-initiation complex during mRNA scanning: Structural role for eIF3c and its control by eIF5.Cell Rep. 2017; 18: 2651-2663Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and interacts with the eIF4F complex. In recent years, a remarkable diversity in the recruitment of eukaryotic ribosomes by mRNAs has been unveiled (13Leppek K. Das R. Barna M. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.Nat. Rev. Mol. Cell Biol. 2018; 19: 158-174Crossref PubMed Scopus (201) Google Scholar, 14Eriani G. Martin F. START: STructure-Assisted RNA translation.RNA Biol. 2018; 15: 1250-1253Crossref PubMed Scopus (4) Google Scholar). This is the case for viruses that have developed simplified systems to improve translation efficiency, allowing also hijacking of the host translation machinery for their own mRNA. Namely, internal ribosomal entry sites (IRESs), located in the 5' untranslated region (5' UTR) of viral mRNA, enable to initiate translation with only a partial set of eIFs in a cap-independent manner sometimes even without any scanning step (1Jackson R.J. Hellen C.U.T. Pestova T.V. The mechanism of eukaryotic translation initiation and principles of its regulation.Nat. Rev. Mol. Cell Biol. 2010; 11: 113-127Crossref PubMed Scopus (1515) Google Scholar, 13Leppek K. Das R. Barna M. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.Nat. Rev. Mol. Cell Biol. 2018; 19: 158-174Crossref PubMed Scopus (201) Google Scholar, 15Bushell M. Sarnow P. Hijacking the translation apparatus by RNA viruses.J. Cell Biol. 2002; 158: 395-399Crossref PubMed Scopus (135) Google Scholar, 16Gross L. Vicens Q. Einhorn E. Noireterre A. Schaeffer L. Kuhn L. Imler J.-L. Eriani G. Meignin C. Martin F. The IRES5′UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure.Nucleic Acids Res. 2017; 45: 8993-9004Crossref PubMed Scopus (19) Google Scholar). Translation initiation of histone H4 mRNA is an alternative initiation mechanism combining canonical (cap dependence) and IRES-like initiation strategies (no-scanning, internal recruitment of initiation factors) (17Martin F. Barends S. Jaeger S. Schaeffer L. Prongidi-Fix L. Eriani G. Cap-assisted internal initiation of translation of histone H4.Mol. Cell. 2011; 41: 197-209Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 18Martin F. Ménétret J.-F. Simonetti A. Myasnikov A.G. Vicens Q. Prongidi-Fix L. Natchiar S.K. Klaholz B.P. Eriani G. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.Nat. Commun. 2016; 7: 12622Crossref PubMed Scopus (24) Google Scholar). H4 mRNA contains specific RNA structures that tether the translation machinery directly on the AUG initiation codon. A double stem-loop structure called eIF4E-sensitive element (4E-SE) binds eIF4E without the need of the cap, and a three-way junction (TWJ) sequesters the m7G cap and facilitates direct 80S ribosomes positioning to the cognate AUG start codon (17Martin F. Barends S. Jaeger S. Schaeffer L. Prongidi-Fix L. Eriani G. Cap-assisted internal initiation of translation of histone H4.Mol. Cell. 2011; 41: 197-209Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The lack of scanning appears to promote high expression levels of histone H4 protein during the S-phase of the cell cycle for rapid incorporation into nucleosomes. The cryo-EM structure of 80S ribosome in complex with H4 mRNA showed that the TWJ forms a repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA) (18Martin F. Ménétret J.-F. Simonetti A. Myasnikov A.G. Vicens Q. Prongidi-Fix L. Natchiar S.K. Klaholz B.P. Eriani G. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.Nat. Commun. 2016; 7: 12622Crossref PubMed Scopus (24) Google Scholar). H4 mRNA harbors a sequence complementary with the h16 loop of the 18S rRNA, which tethers the mRNA to the ribosome to promote proper start codon positioning (18Martin F. Ménétret J.-F. Simonetti A. Myasnikov A.G. Vicens Q. Prongidi-Fix L. Natchiar S.K. Klaholz B.P. Eriani G. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.Nat. Commun. 2016; 7: 12622Crossref PubMed Scopus (24) Google Scholar). This highlights the functional importance of the H4 mRNA structures located in the coding sequence during the initiation process. An additional secondary H4 mRNA structure, also located in the coding sequence, was recently found to interact with eIF3 (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar). The initiation factor eIF3 is capable of selectively recruiting and controlling the expression of several cellular mRNAs by binding to specific stem loops (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar, 20Lee A.S.Y. Kranzusch P.J. Doudna J.A. Cate J.H.D. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.Nature. 2016; 536: 96Crossref PubMed Scopus (134) Google Scholar, 21Pulos-Holmes M.C. Srole D.N. Juarez M.G. Lee A.S.-Y. McSwiggen D.T. Ingolia N.T. Cate J.H. Repression of ferritin light chain translation by human eIF3.Elife. 2019; 8e48193Crossref PubMed Scopus (9) Google Scholar). This regulation occurs primarily through interactions with 5'UTR structural elements, but the role of eIF3 in regulation is not yet clearly established (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar, 22Cate J.H.D. Human eIF3: From "blobology" to biological insight.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20160176Crossref PubMed Scopus (35) Google Scholar), nor is the mechanism by which eIF3 selects its mRNA targets. Composed of 13 subunits (a-m), the structural scaffold of mammalian eIF3 is a multilobed octamer conserved in the proteasome and signalosome complexes (11Valášek L.S. Zeman J. Wagner S. Beznosková P. Pavlíková Z. Mohammad M.P. Hronová V. Herrmannová A. Hashem Y. Gunišová S. Embraced by eIF3: Structural and functional insights into the roles of eIF3 across the translation cycle.Nucleic Acids Res. 2017; 45: 10948-10968Crossref PubMed Scopus (50) Google Scholar, 23des Georges A. Dhote V. Kuhn L. Hellen C.U.T. Pestova T.V. Frank J. Hashem Y. Structure of mammalian eIF3 in the context of the 43S preinitiation complex.Nature. 2015; 525: 491-495Crossref PubMed Scopus (119) Google Scholar, 24Brito Querido J. Sokabe M. Kraatz S. Gordiyenko Y. Skehel J.M. Fraser C.S. Ramakrishnan V. Structure of a human 48 S translational initiation complex.Science. 2020; 369: 1220-1227Crossref PubMed Google Scholar). Six eIF3 subunits (a, c, e, k, l, and m) bear PCI (Proteasome, COP9, eIF3) and two subunits (f, h) bear MPN (Mpr1–Pad1 N-terminal) signature domains. eIF3d seems to be located in a peripheral position, is not required for the integrity of the complex and not conserved across species but is essential in some organisms (23des Georges A. Dhote V. Kuhn L. Hellen C.U.T. Pestova T.V. Frank J. Hashem Y. Structure of mammalian eIF3 in the context of the 43S preinitiation complex.Nature. 2015; 525: 491-495Crossref PubMed Scopus (119) Google Scholar). Near-atomic resolution structure of the human eIF3 in the context of the 48S recently revealed that eIF3d interacts both with the 40S and the octameric core, as well as potentially with eIF3F (24Brito Querido J. Sokabe M. Kraatz S. Gordiyenko Y. Skehel J.M. Fraser C.S. Ramakrishnan V. Structure of a human 48 S translational initiation complex.Science. 2020; 369: 1220-1227Crossref PubMed Google Scholar). eIF3d was also shown to bind the 5' cap of some specific mRNAs in a way reminiscent of eIF4E suggesting the existence of a second mechanism of cap-dependent translation, linked to eIF3d (20Lee A.S.Y. Kranzusch P.J. Doudna J.A. Cate J.H.D. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.Nature. 2016; 536: 96Crossref PubMed Scopus (134) Google Scholar, 25de la Parra C. Ernlund A. Alard A. Ruggles K. Ueberheide B. Schneider R.J. A widespread alternate form of cap-dependent mRNA translation initiation.Nat. Commun. 2018; 9: 3068Crossref PubMed Scopus (43) Google Scholar). Peripheral subunits of eIF3 also include the eIF3b, g, i module that encircles the 40S and connects the mRNA entry channel to the exit site of the ribosome (24Brito Querido J. Sokabe M. Kraatz S. Gordiyenko Y. Skehel J.M. Fraser C.S. Ramakrishnan V. Structure of a human 48 S translational initiation complex.Science. 2020; 369: 1220-1227Crossref PubMed Google Scholar, 26Erzberger J.P. Stengel F. Pellarin R. Zhang S. Schaefer T. Aylett C.H.S. Cimermančič P. Boehringer D. Sali A. Aebersold R. Ban N. Molecular architecture of the 40S⋅eIF1⋅eIF3 translation initiation complex.Cell. 2014; 159: 1227-1228Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 27Llácer J.L. Hussain T. Marler L. Aitken C.E. Thakur A. Lorsch J.R. Hinnebusch A.G. Ramakrishnan V. Conformational differences between open and closed states of the eukaryotic translation initiation complex.Mol. Cell. 2015; 59: 399-412Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Due to the presence of several RNA-binding domains, eIF3 offers multiple opportunities of interactions with its targets. Here we report that eIF3 binds to a stem-loop structure located in the coding region of H4 mRNA downstream of the 4E-SE. Combining cross-linking and ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro, we found that eIF3 interacts with H4 and also with H1, H2A, H2B, and H3 histone mRNAs. We demonstrate a direct interaction of H4 mRNA with eIF3c, d, e and g subunits and suggest the existence of different interaction patterns for the different histone mRNAs. After having inactivated eIF3 in vivo by siRNA interference in G1/S synchronized cells, we selectively monitored histone neosynthesis by [35S] pulse labeling. These experiments reveal that eIF3 could act as a modulator of histones translation particularly in metabolic conditions where eIF3 comes to be limiting. It was previously established that human translation initiation factor eIF3 can target mRNAs in a transcript-specific manner and can function as an activator or repressor of translation (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar, 20Lee A.S.Y. Kranzusch P.J. Doudna J.A. Cate J.H.D. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.Nature. 2016; 536: 96Crossref PubMed Scopus (134) Google Scholar, 21Pulos-Holmes M.C. Srole D.N. Juarez M.G. Lee A.S.-Y. McSwiggen D.T. Ingolia N.T. Cate J.H. Repression of ferritin light chain translation by human eIF3.Elife. 2019; 8e48193Crossref PubMed Scopus (9) Google Scholar). The majority of the mRNAs identified contain a single eIF3-binding site predominantly located within 5'UTR RNA structural elements (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar). By PAR-CLIP a 25 nt H4 mRNA sequence was identified among eIF3 mRNA targets interacting with eIF3 (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar). By contrast, this sequence is located in the coding region of H4 mRNA between nucleotides 294 and 319 (Fig. 1A) in a region adjacent to previously characterized structural elements, namely the TWJ and the 4E-SE (17Martin F. Barends S. Jaeger S. Schaeffer L. Prongidi-Fix L. Eriani G. Cap-assisted internal initiation of translation of histone H4.Mol. Cell. 2011; 41: 197-209Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We determined the secondary structure of H4 mRNA around the potential eIF3-binding site using chemical probing and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) (Fig. 1B). Chemical probing and SHAPE revealed that the potential eIF3-binding site maps to the 3' strand of a 70 nt long stem-loop structure named hereafter eIF3-binding site (eIF3-BS). The primer extension pattern revealed the presence of a large central bulge encompassing nts (261–269 and 299–306) in addition to the two small bulges (ΔG = −28.6 kcal/mol at 37 °C, from nts 250 to 320 (28Zuker M. Mfold web server for nucleic acid folding and hybridization prediction.Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (9474) Google Scholar)). Fourteen nts of the sequence identified by PAR-CLIP (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar) were found in the double-stranded part of the motif (Fig. 1B) in agreement with an interaction of eIF3 occurring in the context of an RNA secondary structure. To evaluate the importance of eIF3-BS, RNA-electrophoretic mobility shift assays were performed using purified full-length H4 mRNA (H4 FL) and three truncated radiolabeled H4 RNA fragments (H4 1–137, H4 137–241, and H4 241–375) generated by in vitro transcription. H4 1 to 137 contains the TWJ, H4 137 to 241 the 4E-SE, and H4 241 to 375 contains the eIF3-BS. Prior to complex formation RNAs were heat denaturated and refolded to promote formation of secondary and tertiary structures. Purified eIF3 complex directly interacted with H4 FL with an estimated Kd of 4 μM. eIF3 moderately interacted with H4 241 to 375 (eIF3-BS) but also with H4 1 to 137 (TWJ) and shifted 39% and 28% of the RNAs respectively at high concentrations of eIF3 (Fig. 1C). In the same conditions only a weak 14% band shift was observed for H4 137 to 241 (Fig. 1C). No retarded complex was obtained in the presence of BSA, used as a negative control. The major eIF3-binding site therefore seems to reside in the eIF3-BS fragment but weaker eIF3 binding can also occur in the H4 1 to 137 fragment, which includes the TWJ. Optimal eIF3 binding therefore seems to require the full-length mRNA. Altogether these results confirm that eIF3 interacts in vitro with the histone H4 mRNA and that the PAR-CLIP defined sequence belongs to the eIF3-BS stem-loop structure. To determine if eIF3 is capable of interacting with all histone mRNAs, we immunoprecipitated eIF3-RNA complexes from HEK293FT cells. Formaldehyde cross-linking was used to stabilize transient interactions and minimize RNP complexes rearrangements (29Hayek H. Gross L. Schaeffer L. Alghoul F. Martin F. Eriani G. Allmang C. Immunoprecipitation methods to isolate messenger ribonucleoprotein complexes (mRNP).in: Vega C. Advanced Technologies for Protein Complex Production and Characterization, Advances in Experimental Medecine and Biology. Vol. 2. Springer, Switzerland2020Google Scholar). The full endogenous eIF3 complex thus stabilized was immunoprecipitated using an antibody directed against the eIF3b subunit (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar, 30Jivotovskaya A.V. Valášek L. Hinnebusch A.G. Nielsen K.H. Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast.Mol. Cell. Biol. 2006; 26: 1355-1372Crossref PubMed Scopus (92) Google Scholar). Western blotting revealed that 11 of the 13 eIF3 subunits (a, b, c, d, e, f, g, h, i, k, l) were specifically co-immunoprecipitated (Fig. 2A). No interaction was detected for GAPDH used as a negative control. The RNAs associated with eIF3 were determined by qRT-PCR (Fig. 2B). The c-JUN mRNA is a target of eIF3 in PAR-CLIP experiments and was used as positive control (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar). The housekeeping, nonhistone mRNAs GAPDH, HPRT, PGK1, ACTB, and LDHA undergo canonical cap-dependent translation (20Lee A.S.Y. Kranzusch P.J. Doudna J.A. Cate J.H.D. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.Nature. 2016; 536: 96Crossref PubMed Scopus (134) Google Scholar). The spliceosomal U2 snRNA was used as a negative control. On average 5% of the housekeeping mRNAs were retained in the anti-eIF3b immunoprecipitation, whereas only 0.2% of U2 snRNA was detected, reflecting the general role of eIF3 in mRNA translation. The relative mRNA enrichment in the eIF3b IP was normalized against that obtained for LDHA mRNA. As expected c-JUN mRNA was co-immunoprecipitated by eIF3 and enriched 4.5 times in the IP compared with LDHA, whereas this is not the case for GAPDH, HPRT, PGK1, and ACTB control mRNAs; this is in accordance with previous results (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar). Our results indicate that all histone mRNAs are significantly enriched in the anti-eIF3b immunoprecipitation. The binding of histone H4 mRNA is the highest with sixfold while histones H1, H2A, H2B, and H3 are enriched between 1.7- and 3-fold (Fig. 2B). On average these results are similar to those observed for c-JUN and confirm that histone mRNAs are prime targets of eIF3. Different modes of interaction between the eIF3 complex and its RNA targets have been established. RNA-binding domains have been identified in the eIF3a, b, and g subunits (31Sun C. Todorovic A. Querol-Audí J. Bai Y. Villa N. Snyder M. Ashchyan J. Lewis C.S. Hartland A. Gradia S. Fraser C.S. Doudna J.A. Nogales E. Cate J.H.D. Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 20473-20478Crossref PubMed Scopus (83) Google Scholar) while eIF3d is capable to bind the cap of several mRNAs by a dedicated cap-binding domain (20Lee A.S.Y. Kranzusch P.J. Doudna J.A. Cate J.H.D. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.Nature. 2016; 536: 96Crossref PubMed Scopus (134) Google Scholar). Only the eIF3a, b, and c subunits bind IRES elements (1Jackson R.J. Hellen C.U.T. Pestova T.V. The mechanism of eukaryotic translation initiation and principles of its regulation.Nat. Rev. Mol. Cell Biol. 2010; 11: 113-127Crossref PubMed Scopus (1515) Google Scholar, 32Cai Q. Todorovic A. Andaya A. Gao J. Leary J.A. Cate J.H.D. Distinct regions of human eIF3 are sufficient for binding to the HCV IRES and the 40S ribosomal subunit.J. Mol. Biol. 2010; 403: 185-196Crossref PubMed Scopus (27) Google Scholar, 33Ujino S. Nishitsuji H. Sugiyama R. Suzuki H. Hishiki T. Sugiyama K. Shimotohno K. Takaku H. The interaction between human initiation factor eIF3 subunit c and heat-shock protein 90: A necessary factor for translation mediated by the hepatitis C virus internal ribosome entry site.Virus Res. 2012; 163: 390-395Crossref PubMed Scopus (15) Google Scholar) while eIF3 mRNA targets identified by PAR-CLIP interact with distinct combinations of the eIF3a, b, d, and g subunits (19Lee A.S.Y. Kranzusch P.J. Cate J.H.D. eIF3 targets cell proliferation mRNAs for translational activation or repression.Nature. 2015; 522: 111-114Crossref PubMed Scopus (178) Google Scholar). In order to precisely identify the subunits of the eIF3 complex in direct interaction with the H4 mRNA, we performed UV cross-linking experiments using a uniformly radiolabeled ThioU-H4 mRNA transcript in the presence of purified eIF3 complex (Fig. 3). After RNase A digestion, only radioactive mRNA fragments protected against degradation because of their interaction with eIF3 remained cross-linked to eIF3 subunits. Separation of the cross-linked products by denaturing gel electrophoresis revealed radiolabeling of at least four different eIF3 subunits with apparent molecular weights of 110, 65, 50, and 45 kDa (Fig. 3A). Several eIF3 subunits share similar molecular weight. This is the case for the subunits eIF3a, b, and c (110 kDa), eIF3d and l (65 kDa) as well as eIF3e, f, and g (45 kDa). To identify the radiolabeled eIF3 subunits, cross-linked products were separated by two-dimensional gel electrophoresis (2D-gel) (Fig. 3B) followed by western blot analysis using antibodies directed against 11 of the 13 eIF3 subunits (Fig. 3, C and D). Our results show cross-linking signals between the subunits eIF3c, d, e, and g (Fig. 3C) and H4 mRNA for which radioactivity and western blot signals overlay. eIF3 subunits undergo numerous posttranslational modifications (34Damoc E. Fraser C.S. Zhou M. Videler H. Mayeur G.L. Hershey J.W.B. Doudna J.A. Robinson C.V. Leary J.A. Structural characterization of the human eukaryotic initiation factor 3 protein complex by mass spectrometry.Mol. Cell. Proteomics. 2007; 6: 1135-1146Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), this is reflected by the dotted migration profile on the 2D-gel, each dot corresponding to the different levels of modifications, and thus isoelectric charge of the protein (Fig. 3, C–E). The migration profile of the proteins is partially shifted upon cross-linking and spreads over a wide range of pH due to the presence of the additional charges coming from the cross-linked RNA moiety (Fig. 3E). By contrast, the western blot signals of the eIF3 subunits b, f, h, i, k do not overlap with the radioactivity signals, showing that they do not interact with H4 mRNA (Fig. 3D). The four subunits eIF3c, d, e, and g that we have identified in interaction with H4 mRNA play important roles in the formation and positioning of the eIF3 complex in the 80S ribosome. eIF3c and e subunits belong to the structural core of eIF3. Only eIF3g has an RNA Recognition Motif (RRM) (35ElAntak L. Tzakos A.G. Locker N. Lukavsky P.J. Structure of eIF3b RNA recognition motif and its interaction with eIF3j: Structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit.J. Biol. Ch
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