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. 2021 Jul 2;10:e66493. doi: 10.7554/eLife.66493

Figure 2. Increased PABPC promotes translation of endogenous short-tailed mRNAs, thereby diminishing coupling between tail length and TE.

(A) The effect of PABPC on coupling between tail length and TE in frog oocytes. Shown is the relationship between TE and median poly(A)-tail length in oocytes injected with either water or mRNAs encoding either eGFP, PABPC1, or ePAB (injecting 16 fmol mRNA per oocyte). Results are shown for mRNAs from genes with ≥100 poly(A) tags. Each poly(A) tag represents a pair of sequencing reads that identify the mRNA and its poly(A) tail length. Rs is the Spearman’s correlation coefficient. (B) A global increase in TE observed upon overexpressing ePAB. TE in ePAB-overexpressing oocytes is compared with TE in eGFP-expressing oocytes. Experiment was as in A, except rRNAs were not depleted, and TE values are normalized to those of mitochondrial mRNAs. Also shown are results from a co-injected short-tailed Nluc reporter and a long-tailed Fluc reporter. (C) Effect of expressing PABPC1 on protein synthesis in oocytes. At the left is a gel showing total protein after injection of either water or the indicated mRNA (injecting 4 fmol mRNA per oocyte), with or without treatment with cycloheximide (CHX), as visualized by Coomassie staining. At the right is the same gel showing protein synthesis, as visualized by incorporation of 35S-methionine and 35S-cysteine. Prominent bands presumably represent PABPC1, eGFP, and a not fully denatured form of eGFP (asterisk) expressed from injected mRNAs. (D) Quantification of the effect of expressing PABPC1 on protein synthesis in oocytes, as measured in (C) and two additional biological replicates. Only regions above the PABPC1 band, and between the PABPC1 band and the top eGFP band, were used for quantification. Values were normalized to that of the mean value from water-injected oocytes (error bars, standard deviation; p values, one-sided t-tests; n.s., not significant). (E) The preferential effect of overexpressing ePAB on the TE of short-tailed mRNAs. TE fold changes observed between ePAB-overexpressing and eGFP-expressing oocytes are plotted as a function of median tail length in eGFP-expressing oocytes. TE and tail-length values were obtained from different batches of oocytes; results are shown for mRNAs from genes with ≥100 poly(A) tags. TE values are normalized to those of mitochondrial mRNAs. (F) Effect of overexpressing ePAB on the distribution of TE values observed in frog oocytes. Shown is the TE distribution observed in ePAB-overexpressing oocytes and that observed in eGFP-expressing oocytes. Tail-length measurements in this figure were obtained using TAIL-seq.

Figure 2—source data 1. Source data for values shown in Figure 2D.

Figure 2.

Figure 2—figure supplement 1. Increased PABPC increases translation of endogenous short-tailed mRNAs in frog oocytes.

Figure 2—figure supplement 1.

(A) Increased relative translation of short-tailed mRNAs after overexpressing PABPC in oocytes. Shown for each gene with ≥100 poly(A) tags is the change in relative TE observed after overexpressing either eGFP, PABPC1 or ePAB, plotted as a function of median poly(A)-tail length in water-injected oocytes. (B) Poor correspondence between TE changes and tail-length changes after overexpressing PABPC in oocytes. TE changes of (A) are plotted as a function of median poly(A) tail-length changes. (C) Depletion of frog mitochondrial mRNAs by the Illumina Ribo-Zero Gold kit. Tail-length measurements in this figure were obtained using TAIL-seq.