eIF3 interacts with histone H4 messenger RNA to regulate its translation

In eukaryotes, various alternative translation initiation mechanisms have been unveiled for the of specific mRNAs. Some do not conform to conventional scanning-initiation model. Translation histone H4 mRNA combines both canonical (cap-dependent) and viral strategies (no-scanning, internal recruitment factors). Specific structures tether machinery directly onto codon allow massive production during S phase cell cycle. The human eukaryotic factor 3 (eIF3), composed 13 subunits (a-m), was shown selectively recruit control expression several cellular Whether eIF3 mediates remains be elucidated. Here, we report that binds a stem-loop structure (eIF3-BS) located in coding region mRNA. Combining cross-linking ribonucleoprotein immunoprecipitation experiments vivo vitro, also found H1, H2A, H2B, H3 We identified direct contacts between eIF3c, d, e, g subunits, mRNAs but observed distinct interaction patterns each Our results show depletion reduces binding modulates neosynthesis, suggesting synthesis histones is sensitive levels eIF3. Thus, provide evidence acts as regulator translation. requires multiple complexes factors (eIFs) assemble elongation-competent ribosomes (1Jackson R.J. Hellen C.U.T. Pestova T.V. mechanism principles its regulation.Nat. Rev. Mol. Cell Biol. 2010; 11: 113-127Crossref PubMed Scopus (1515) Google Scholar, 2Valášek L.S. ‘Ribozoomin’ – from perspective ribosome-bound (eIFs).Curr. Protein Pept. Sci. 2012; 13: 305-330Crossref (86) Scholar). recognition m7G cap by eIF4E-binding part complex eIF4F (composed three eIF4E, eIF4A, eIF4G) constitutes first step prerequisite ribosomal attachment (3Fortes P. Inada T. Preiss Hentze M.W. Mattaj I.W. 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It previously target transcript-specific function activator repressor majority contain single eIF3-binding predominantly within elements By PAR-CLIP 25 nt among interacting contrast, this nucleotides 294 319 (Fig. 1A) adjacent characterized namely 4E-SE determined around potential using chemical probing selective 2’-hydroxyl acylation analyzed primer extension (SHAPE) 1B). Chemical SHAPE maps 3’ strand 70 long named hereafter (eIF3-BS). pattern large central bulge encompassing nts (261–269 299–306) addition small bulges (?G = ?28.6 kcal/mol 37 °C, 250 320 (28Zuker Mfold web server nucleic acid folding hybridization prediction.Nucleic 2003; 3406-3415Crossref (9474) Scholar)). Fourteen were double-stranded motif 1B) agreement occurring structure. To evaluate eIF3-BS, RNA-electrophoretic mobility shift assays performed purified full-length (H4 FL) truncated radiolabeled fragments 1–137, 137–241, 241–375) generated vitro transcription. 1 137 TWJ, 241 4E-SE, 375 eIF3-BS. Prior formation RNAs heat denaturated refolded tertiary structures. Purified interacted FL estimated Kd 4 ?M. moderately shifted 39% 28% respectively concentrations 1C). same weak 14% band No retarded obtained BSA, used negative control. major therefore reside eIF3-BS fragment weaker occur fragment, includes TWJ. Optimal require Altogether these confirm defined belongs determine if all mRNAs, immunoprecipitated eIF3-RNA HEK293FT cells. Formaldehyde stabilize transient minimize RNP rearrangements (29Hayek Gross Alghoul Allmang Immunoprecipitation methods isolate messenger (mRNP).in: Vega Advanced Technologies Complex Production Characterization, Advances Experimental Medecine Biology. Vol. 2. Springer, Switzerland2020Google full endogenous thus stabilized antibody directed against eIF3b 30Jivotovskaya A.V. Nielsen K.H. eIF2 independently yeast.Mol. 26: 1355-1372Crossref (92) Western blotting 11 b, f, h, i, l) specifically co-immunoprecipitated 2A). detected GAPDH associated qRT-PCR 2B). c-JUN positive housekeeping, nonhistone GAPDH, HPRT, PGK1, ACTB, LDHA undergo spliceosomal U2 snRNA On average 5% housekeeping retained anti-eIF3b immunoprecipitation, whereas 0.2% detected, reflecting general relative enrichment IP normalized As expected enriched 4.5 times compared LDHA, ACTB mRNAs; accordance previous indicate are significantly immunoprecipitation. highest sixfold while 1.7- 3-fold similar those prime Different modes established. domains eIF3a, (31Sun Todorovic Querol-Audí Bai Villa Snyder Ashchyan Hartland Gradia Nogales reconstitution (eIF3).Proc. Natl. Acad. U. 108: 20473-20478Crossref (83) dedicated Only c 32Cai Andaya Gao Leary Distinct regions sufficient HCV subunit.J. 403: 185-196Crossref 33Ujino Nishitsuji Sugiyama Suzuki Hishiki Shimotohno Takaku heat-shock 90: necessary mediated hepatitis C site.Virus 163: 390-395Crossref (15) combinations order precisely identify UV uniformly ThioU-H4 transcript 3). RNase digestion, radioactive protected degradation because remained cross-linked subunits. Separation products denaturing gel electrophoresis radiolabeling least four apparent molecular weights 110, 65, 50, 45 kDa 3A). Several share weight. (110 kDa), l (65 kDa) eIF3e, (45 kDa). separated two-dimensional (2D-gel) 3B) followed western blot analysis antibodies 3, D). signals 3C) radioactivity overlay. numerous posttranslational modifications (34Damoc Videler Mayeur G.L. Hershey J.W.B. Robinson C.V. mass spectrometry.Mol. Proteomics. 2007; 1135-1146Abstract (107) reflected dotted migration profile 2D-gel, dot corresponding modifications, isoelectric charge C–E). proteins partially upon spreads over wide range pH due charges coming moiety 3E). k overlap signals, showing they 3D). play important ribosome. belong core eIF3g Recognition Motif (RRM) (35ElAntak Tzakos Locker Lukavsky eIF3j: 40 Ch