Translation initiation factor eIF3 promotes programmed stop codon readthrough

Abstract

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

Figure 1.

Readthrough measurements using the standard dual luciferase assay are not sensitive to defects in translation initiation. (A) Schematics of the standard dual luciferase readthrough reporter constructs with variable stop codons (or a CAA coding codon) under the P_PGK promoter adapted from (29). (B) Schematics of the modified dual luciferase readthrough reporter constructs containing the HiPV IRES in their 5′ UTRs. (C) eIF3 mutations affect stop codon readthrough independently of their initiation phenotypes. The TIF35 wt, tif35-KLF and tif35-C121R mutant alleles were introduced into the H464 strain by plasmid shuffling. The resulting transformants were grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs schematically illustrated in panel A as described in Materials and Methods. (D) Same as in panel C except that modified dual luciferase reporter constructs YEp-HiPV-UGAC-L and YEp-HiPV-CAAC-L, schematically illustrated in panel B, were employed.

Figure 2.

Wild-type eIF3 promotes stop codon readthrough but only on stop codons in the unfavorable termination context. The TIF35 wt and tif35-KLF mutant alleles were introduced into the PBH140 (tif35Δ SUP35) and PBH134 (tif35Δ sup35-N536T) strains by plasmid shuffling. The resulting transformants were grown in SD and processed for stop codon readthrough measurements as described in Materials and Methods. Percentages of readthrough of tif35-KLF given in the table were calculated from measured values (mean ± SE; n = 6) normalized to TIF35 wt. Changes in readthrough levels were analyzed by the student's t-test and shown to be statistically significant only for those values marked with the asterisk (P < 0.01).

Figure 3.

eIF3 stimulation of stop codon readthrough is dependent on its association with pre-termination 80S complexes in vivo. (A) Changes in amounts of translation factors associated with the pre-TCs in cells bearing TIF32 mutations in vivo. Plasmid-born mutant alleles of TIF32 (tif32-Box6, tif32-Δ8 and tif32-Box17) and its corresponding wild-type (TIF32) were introduced into YBS52 strain by plasmid shuffling. The resulting transformants were grown in SD medium at 30°C to an OD₆₀₀ of ∼1 and cross-linked with 0.5% HCHO prior to harvesting. WCEs were prepared, separated on a 5–45% sucrose gradient by centrifugation at 39 000 rpm for 2.5 h and heavier polysome fractions were collected and treated with RNase I to separate the pre-initiation complexes (PICs) from 80S couples on actively translated mRNAs. Thus treated samples were subjected to the second sucrose gradient centrifugation according to the resedimentation protocol described before and 80S couples—predominantly containing termination complexes (18)—were collected, loaded in six two-fold dilutions onto the SDS-PAGE gel and processed for western blot analysis with antibodies raised against factors shown above the strips. Three representative dilutions for each factor are shown. (B) Quantification of selected factors from at least three independent experiments shown in Figure 3A with corresponding P-values, where applicable. Western blot signals from each of the six two-fold dilutions obtained with individual anti-bodies were quantified by NIH ImageJ and plotted against their corresponding loadings. Individual slopes (representing relative amount of each factor in mutant cells) calculated from the linear regression of resulting plots were normalized to the slope obtained with the TIF32 wt strain, which was set to 100%. (C) Changes in amounts of translation factors associated with the pre-TCs in cells bearing the tif35-KLF mutation in vivo. Quantification of selected factors from at least three independent experiments carried out as described in panel A with the exception that tif35-KLF and its corresponding TIF35 wt were introduced into the H464 strain by plasmid shuffling.

Figure 4.

eIF3 is one of the major players in programmed stop codon readthrough. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles (generated as described in Figure 2) were grown and processed for stop codon readthrough measurements as described in Materials and Methods, except that besides our standard (‘control’) pTH477 readthrough construct (shown in Figure 1A), five derivatives with variable hexanucleotide sequence immediately following the stop codon were also employed: PBB85 (for SUP45), PBB83 (for ADE1), PBB84 (for PDE2), PBB80 (for BSC4) and PBB82 (for TMV).

Figure 5.

eIF3 interferes with decoding of the third position of the stop codon allowing incorporation of near-cognate tRNAs with the mismatch at the same position. (A) eIF3 promotes incorporation of both existing near-cognate tRNAs with a mismatch at the third position of the UGA stop codon. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles (generated as described in Figure 2) were transformed with either empty vector (EV), high copy (hc) tW(CCA)G1 or hc tC(GCA)P1 and the resulting transformants were grown and processed for stop codon readthrough measurements as described in Materials and Methods. (B) eIF3 enhances incorporation of the near-cognate tRNA also at the UAA stop codon with a mismatch at the third position [tY(GUA)J2—encoding tyrosine] but not with a mismatch at the second position [tY*(UCA)J2] or non-cognate [tW(CCA)G1—encoding tryptophan]. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles (generated as described in Figure 2) were transformed with empty vector (EV), hc tY(GUA)J2, hc tY*(UCA)J2 or hc tW(CCA)G1 and subsequently also with the readthrough construct YEp-R/T-UAAC-L and the resulting transformants were grown and processed for stop codon readthrough measurements as described in Materials and Methods. (C) eIF3 does not affect incorporation of both existing near-cognate tRNAs with a mismatch at the first position of the UGA stop codon. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles (generated as described in Figure 2) were transformed with either empty vector (EV), hc tR(UCU)E or hc tG(UCC)O and the resulting transformants were grown and processed for stop codon readthrough measurements as described in Experimental Procedures. (D) eIF3 impedes stop codon decoding by fully cognate tRNA. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles (generated as described in Figure 2) were transformed with either empty vector (EV) or a single copy (sc) plasmid carrying tY*(UCA)J2 and the resulting transformants were grown and processed for stop codon readthrough measurements as described in Materials and Methods. (E) Paromomycin nullifies the effect of wt eIF3 on programmed stop codon readthrough. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles (generated as described in Figure 2) were grown in SD without or with 200μg/ml paromomycin for 6 h and processed for stop codon readthrough measurements as described in Materials and Methods.

Figure 6.

Human eIF3 promotes stop codon readthrough. HeLa cells were transfected with siRNA against the eIF3a and eIF3g subunits (eIF3a^K.D. and eIF3g^K.D.) or non-targeting siRNA (nt) as control. Two days later, the siRNA-treated cells were further transfected with the bicistronic firefly/renilla luciferase readthrough reporters (40). Both luciferases are separated by a stop (UAG) or sense codon (CAG) followed—in both cases—by a highly readthrough permissive sequence (CAAUUA) from TMV (41). The UAG-C readthrough is displayed as % of the signal obtained with the sense codon reporter; standard deviations are given.

Figure 7.

Model: translation initiation factor eIF3 promotes programmed stop codon readthrough. (A) Canonical termination; stop codon in the termination favorable context appears in the A-site (only UAG and UAA stop codons are indicated for illustration purposes; UGA works by the same mechanism), eRF1 in complex with eRF3.GTP binds to it and samples the codon in a two-step process including conformational re-arrangements of the eRF1-NTD. During the second step the ribosome by itself co-participates in this accommodation phase that ultimately leads to GTP hydrolysis on eRF3, polypeptide release and ribosomal recycling (see text for further details). (B) Programmed stop codon readthrough; stop codon occurs in the unfavorable termination context bearing specific consensus sequences like CAR-NBA in its 3′ UTR—in this particular case proposed to base-pair with 18S rRNA. The eIF3 presence in the pre-TC—perhaps in co-operation with these sequences—alters the decoding property of the nucleotide at the third stop codon position. This prevents its proper decoding during the second sampling step and subsequently, after the eRF1-eRF3.GTP complex rejection, allows incorporation of near-cognate tRNAs with the mismatch at the third position to read through the stop codon and continue with elongation.