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Initiation Context Modulates Autoregulation of Eukaryotic Translation Initiation Factor 1 (Eif1)

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Initiation Context Modulates Autoregulation of Eukaryotic Translation Initiation Factor 1 (Eif1) Initiation context modulates autoregulation of eukaryotic translation initiation factor 1 (eIF1) Ivaylo P. Ivanova,b,1,2, Gary Loughrana,1, Matthew S. Sachsc, and John F. Atkinsa,b,2 aBioSciences Institute, University College Cork, Cork, Ireland; bDepartment of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330; and cDepartment of Biology, Texas A&M University, College Station, TX 77843 Edited by Jonathan S. Weissman, University of California, San Francisco, CA, and approved September 20, 2010 (received for review June 30, 2010) The central feature of standard eukaryotic translation initiation is Autoregulation of the synthesis of translation factors is often small ribosome subunit loading at the 5′ cap followed by its 5′ to 3′ observed (17). Initiation of IF3 translation begins with an AUU scanning for a start codon. The preferred start is an AUG codon in an codon that is an important component of a negative feedback optimal context. Elaborate cellular machinery exists to ensure the regulatory loop. High levels of IF3 protein increase discrimination fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) against initiation at its AUU start codon, which leads to reduced plays a central role in this process. Here we show that the translation synthesis of the protein (18, 19). The present work addresses of eIF1 homologs in eukaryotes from diverse taxa involves initiation whether a counterpart autoregulatory mechanism controls initia- from an AUG codon in a poor context. Using human eIF1 as a model, tion at the eIF1 start codon. we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own Results translation, establishing that variability in the stringency of start co- The Initiation Codon of eIF1 in Diverse Eukaryotes Has Poor Initiation don selection is used for gene regulation in eukaryotes. We show that Context. To investigate whether eIF1 might be subject to autor- the stringency of start codon selection (preferential utilization of op- egulation similar to that of IF3 in bacteria, the initiation site of timal start sites) is increased to a surprising degree by overexpress- eIF1 was analyzed. Sequences from more than 500 eIF1 homologs ing eIF1. The capacity for the cellular level of eIF1 to impact initiation were assembled and compared. eIF1 ORFs are relatively short through the variable stringency of initiation codon selection likely (110–120 codons). The majority have a single AUG initiation co- fi has signi cant consequences for the proteome in eukaryotes. don and no other downstream in-frame AUG. A minority have one or more additional in-frame AUG codons present in the Kozak context | non-AUG | IF3 second half of the ORF that are unlikely to initiate translation of a functional protein. Examination of the eIF1 sequences precluded ranslation initiation in eukaryotes is substantially different the possibility of non-AUG initiation for eIF1 that would be Tfrom that in bacteria. The standard mode involves scanning of analogous to the AUU initiation for bacterial IF3. The data in- a complex involving the small ribosomal subunit from the 5′ cap to dicate that the first in-frame AUG is the sole initiation codon the initiation codon. There is limited reinitiation following trans- available for translation of the eIF1-coding region. lation of a coding sequence (1, 2). Genetic and biochemical studies The context of the eIF1 AUG initiator revealed a startling have revealed several eukaryotic factors involved in selecting the conserved pattern that deviates dramatically from the optimal correct initiation codon (3–6). Further analyses pointed toward consensus. In almost every case, the initiation context appears to eukaryotic initiation factor 1 (eIF1) as the key mediator of this be very poor. Of 233 metazoan eIF1 sequences examined, 217 process (7–10). eIF1 binds near the P-site of the small ribosomal (93%) lack both a purine at −3 and a G at +4 (Fig. 1A and Fig. subunit (11); this binding is thought to lead to an open confor- S1A); this is also the case for 88 of 91 (97%) fungal sequences mation of the preinitiation complex favoring scanning (12, 13). examined (Fig. 1B and Fig. S1B) and for 220 eIF1 sequences (Fig. Release of eIF1 leads to formation of a closed conformation fa- 1C and Fig. S1C) from the plant kingdom examined. The disparity voring initiation; dissociation of eIF1 from the preinitiation com- between eIF1 context and preferred context is also observed when fi plex is the commitment step in start-codon selection (4). Both in the speci c contexts for each individual species are compared (Fig. fl vitro and in vivo experiments have shown that eIF1 is crucial for S2). We found that some owering plants have an additional eIF1 discrimination between initiation at AUG or near-cognate non- paralog, which is apparently under relaxed selective pressure and AUG codons (4, 6–8). likely has a nonredundant function. Its sequence has a conserved Work accomplished nearly 30 y ago showed that, in mammals, +4 G (Fig. S3). During the preparation of this manuscript, another strong bias exists for the six nucleotides preceding the preferred group reported similar conservation of poor initiation context on initiation AUG and the nucleotide following it (14). Subsequently, the basis of an analysis of seven eIF1 eukaryotic homologs (20). it was demonstrated that sequences in this region determine the Humans have two paralogs of eIF1 (EIF1 and EIF1B); both efficiency of initiation at an AUG (15). The sequence GCC(A/G) have poor initiation contexts. We used human EIF1 as a repre- sentative example to study the potential translational regulation of CCAUGG, referred to as the Kozak consensus, is optimal for ini- eIF1. The human EIF1 initiation site is UAUCGUAUGUCC, tiation; the underlined nucleotides at positions −3 and +4 (relative which is identical to the metazoan eIF1 consensus (Fig. 1A). To to the +1 A of AUG, shown in italics) are most important for effi- cient initiation (15). In vitro, eIF1 is crucial for discriminating between AUG codons Author contributions: I.P.I. and G.L. designed research; G.L. performed research; I.P.I., G.L., in poor and good initiation contexts and also for discriminating M.S.S., and J.F.A. analyzed data; and I.P.I., G.L., M.S.S., and J.F.A. wrote the paper. ′ against AUGs that are too close to the mRNA 5 -end (7). Bacterial The authors declare no conflict of interest. initiation factor 3 (IF3) is a functional analog of eIF1 in that, like This article is a PNAS Direct Submission. eIF1, it has the ability to discriminate between initiation at AUG 1I.P.I. and G.L. contributed equally to this work. and near-cognate non-AUG codons. Even though they are not 2 ’ To whom correspondence may be addressed. E-mail: [email protected] or homologs, eIF1 and IF3 can functionally substitute each others [email protected]. ability to dissociate aberrant initiation complexes in the heterol- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ogous system (16). 1073/pnas.1009269107/-/DCSupplemental. 18056–18060 | PNAS | October 19, 2010 | vol. 107 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1009269107 Downloaded by guest on September 28, 2021 AU A U G A AC A U A U AG C U UC G C AC A A U AG U AG A C CC GA G G UGU G C C UC AU G good context good* context -8 -9 -8 -7 -6 -5 -4 -3 -2 -1 +4 +5 +6 GCCACCAUGG GCCACCAUGU UA C B GC C AU U C 5‘ UAUCGUAUGU 3‘ C AG G U U A U U eIF1 U GC U A C A AUG G A C G C A G GAGA UG WT eIF1 context -8 -9 -8 -7 -6 -5 -4 -3 -2 -1 AU G +4 +5 +6 C C CA GU U U A G A CG A C U U U U C AG G GG UA A C G U -8 -9 -8 -7 -6 -5 -4 U-3 U-2 C-1 A U G U+4 C+5 +6 5‘ UAUCGUAUGU Firefly luciferase 3‘ WT eIF1 context FFLuc/RLuc D 15 25 0 5 10 20 5‘ GCCACCAUGG Renilla luciferase 3‘ good context 25.9% UAGGCCACCAUGGCC UAGUAUCGUAUGUCC (best context) (eIF1 WT context) Fig. 1. Starting codon context of eIF1 genes is poor, which leads to in- eIF1 WT eIF1 good eIF1 good* N/A eIF1 used efficient initiation. (A) Logo representation of the initiation context of 233 B 10x 1x 10x 1x 10x 1x N/A amount DNA metazoan eIF1 homologs. (B) Representation of the initiation context of 91 kDa fungal eIF1 homologs. (C) Representation of the initiation context of 220 15 Anti-eIF1 plant eIF1 homologs (for discussion of excluded plant paralogs, see legend for Fig. S3). Letter heights are proportional to the frequency of conservation. 50 Anti-β actin The consensus conservation is shown at the top of each logo; the position 37 relative to the A of the AUG start codon is indicated below (crucial positions 1 2 3 4 5 6 7 −3 and +4 are in red). Alignments of the sequences used to generate the 0.04 fi C logogram are shown in Fig. S1.(D) Relative initiation ef ciency of consensus 0.035 mammalian initiation context and human EIF1 initiation context (arbitrary 0.03 units). The results are derived from dual luciferase transfection experiments FFLuc/RLuc as described in Materials and Methods. 0.025 0.02 2.1x 2.4x 0.015 investigate the translation initiation potential of different codons 3.6x 4.9x in different sequence contexts, sequences were positioned to en- 0.01 8.6x able the possibility of initiating translation of a firefly luciferase 0.005 11.8x reporter in a vector that was transfected into HEK-293T cells.
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