Cellular translational enhancer elements that recruit eukaryotic initiation factor 3

Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5′-untranslated regions (5′-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5′-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5′-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation programs.

UTRs are sufficient to drive greater than hundred-fold differences in translation initiation  Duran and Gilbert, 2012). Despite great progress towards illuminating the fundamental 34 mechanisms by which eukaryotic mRNAs recruit ribosomes to initiate translation, many 35 quantitatively large mRNA-specific differences in translation initiation remain unexplained.

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For most cellular messages, translation initiation requires the concerted action of many 38 eukaryotic initiation factors (eIFs). Cellular mRNAs begin with a 5′-m 7 G cap that is recognized 39 by the eIF4E subunit of the cap binding complex, eIF4F. Ribosomes are recruited to mRNA as

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A high-throughput assay for direct binding of eIF3 to specific 5′-UTRs 83 Because of the emerging role of eIF3 in mediating the translation of specific transcripts, we asked 84 whether specific yeast mRNAs bind eIF3 with high affinity. Such direct binding to eIF3 could   (Lambert et al., 2014). 5′-UTRs were defined as "bound" or 128 "not bound" at each concentration of eIF3 using a standard deviation-like cutoff, enrichment > (1 129 +range 33 rd to 66 th percentile), as previously described (Taliaferro et al., 2016). 5′-UTRs that 130 bound non-specifically to nitrocellulose in the absence of eIF3 were eliminated from 131 consideration ( Figure S1d). Overall, we identified 1164 5′-UTRs bound in two or more adjacent  Table 1).

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Representative 5′-UTRs, which included eIF3 binders and non-binders, were tested by 136 nitrocellulose filter binding with purified eIF3 at concentrations ranging from 15nM to 1M to 137 validate the results from RBNS and determine the K d of binding (Methods). Out of eleven tested 138 binders, nine bound to eIF3 with K d values from 100 to 250 nM and two did not bind tightly (K d 139 > 1000 nM) (Figures 2 and S2a-g). In parallel, we tested five non-binding 5′-UTRs (enrichment < 140 1.59 in 166 nM library) from the RBNS assay, all of which bound weakly or not at all with  Bound 5′-UTRs are enriched in AMAYAA motifs in unstructured regions 156 Yeast eIF3 is composed of five distinct subunits, several of which contain known or potential sequences identified in each (Figures 3a and S3a). Therefore, we focused further analysis on the 167 AMAYAA sequence.

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If the AMAYAA motif influences eIF3 binding, we reasoned that mRNAs containing 170 more copies of this motif would display higher affinities for eIF3. We counted the motifs in each 171 RNA and divided the pool based on the number of motifs in the sequence. We observed that the 172 presence of the AMAYAA motif in the RNA significantly increases enrichment score in a dose-173 dependent manner for sequences with 0, 1, 2, or 3+ motifs (p<1.5e-5, Figure 3b). This behavior is 174 similar to other RNA binding proteins with known binding motifs which were analyzed using    of ribosome-protected footprints normalized to total mRNA levels, is a measure of translation activity that is thought to be predominantly affected by mRNA-specific differences in translation 219 initiation (Shah et al., 2013). We therefore compared ribosome density in exponentially growing 220 yeast to eIF3 binding as approximated by enrichment in the 166 nM library. We restricted this 221 analysis to 2469 genes with a dominant 5′-UTR isoform (Methods) because ribosome footprints 222 observed within CDS regions cannot be assigned to a specific 5′-UTR isoform and many 223 alternative 5′-UTR isoforms show differential binding to eIF3. In fact, 913 tested genes have 224 isoforms with significantly different (p adj < 0.05) eIF3 enrichment ( Figure S4a, Supplemental 225 Table 2).

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The mRNAs with 5′-UTRs that bound eIF3 in vitro ( Figure 1e binding on translation initiation for many genes.

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Next, we asked whether mRNAs with 5′-UTRs that preferentially bound eIF3 in vitro are 236 able to maintain translation under conditions where eIF3 is limiting. We performed ribosome 237 profiling in tif32-td prt1-td cells in which eIF3 levels were substantially depleted and bulk   Figure S4g).

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We hypothesized that upstream eIF3 binding motifs enhance translation of uORFs which 293 leads to fewer ribosomes initiating translation of the main ORF. Additionally, as ribosomes sense 294 the 5′-UTR sequence by scanning, we hypothesized that the order of the motif and the uORF on 295 the 5′-UTR will matter. Therefore, we examined ribosome density on 78 mRNAs that contain one    Our results raise the question of which parts of the multi-protein eIF3 complex are 366 responsible for high-affinity binding to specific cellular 5′-UTRs. It is likely that distinct eIF3  The comprehensive approach used here to identify cellular 5′-UTRs that bind to wild-type   Figure S1: Control experiments for eIF3 RNA Bind-n-Seq a) Commassie blue-stained tris-407 glycine gel of purified eIF3 with indicated subunits. The major contaminant observed is eIF5. b) 408 Purified eIF3 restores translational activity to heat-inactivated extracts from prt1-1 mutant yeast.

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Reported are the relative luciferase activities of eIF3 heat sensitive (prt1-1) and isogenic (PRT1)    The AMAYAA motif is associated with increased ribosome density in 5′-UTRs without uORFs.

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In 5′-UTRs with multiple uORFs, the AMAYAA motif is associated with decreased ribosome 452 density. b, c, f, g) Depicted are the log 2 -transformed values of ribosome densities.

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Yeast strains and growth 458 Genotypes are listed in Table I. Strain LPY87 was grown for eIF3 purification as previously  . When a 5′-UTR started within 10 nts of its nearest neighbor, the 505 sequences were merged. Inclusion in the pool also required the following: 5′-UTRs must be 506 expressed within 25% of the mode abundance for a given 5′ UTR, and 5′-UTRs must make up at 507 least 5% of the total abundance for that ORF, unless the mode was <5% of the total, in which 508 case we used the mode. Upstream AUGs within 761 (6.3% of all) 5′-UTR sequences were 509 mutated to AGT such that the first AUG encountered by a scanning pre-initiation complex  Each strain and its matching wild type (Table I)