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

Figure 1. PABPC overexpression uncouples poly(A)-tail length and TE in frog oocytes.

(A) Schematic of capped T7 transcripts with two different tail lengths, which were used as reporter mRNAs. Additional sequences beyond the HDV sequence are not depicted. (B) The effect of tail length on relative yields of in vitro translation of short- and long-tailed Nluc reporter mRNAs, in either frog oocyte extract (left) or rabbit reticulocyte lysate (right). The number above each bracket indicates the fold difference of the mean normalized luciferase signal (error bars, standard deviation from three technical replicates). (C) The effect of purified PABPC1 on relative yields of in vitro translation of short- and long-tailed Nluc reporter mRNAs in frog oocyte extract. Purified eGFP and PABPC1 were each added as indicated. Otherwise, this panel is as in (B). (D) Experimental scheme for serial-injection of mRNAs into frog oocytes. (E) The effect of overexpressing PAPBC1 and a PABPC1 M161A mutant on relative translation of Nluc reporter mRNAs in frog oocytes. Differential PABPC1 expression was achieved by injecting the indicated amount of mRNA in the first injection (error bars, standard deviation from three biological replicates). Otherwise, this panel is as in (B). (F) The effect of PAPBC1 overexpression on translation of reporter mRNAs with different 3′-end structures in frog oocytes. Shown are raw luciferase yields from Nluc reporters that have either a short poly(A) tail, a long poly(A) tail, a histone mRNA 3′-end stem-loop, or a Malat1 triple-helix 3′-end in oocytes overexpressing either eGFP or PABPC1 (error bars, standard deviations from three biological replicates). p values are from one-sided t-tests (n.s., not significant). For overexpression, 2.4 fmol mRNA was injected per oocyte.

Figure 1—source data 1. Source data for luciferase values shown in Figure 1 and Figure 1—figure supplement 2.

Figure 1.

Figure 1—figure supplement 1. Supporting data for reporter experiments examining the effect of PAPBC levels on coupling between tail length and translation.

Figure 1—figure supplement 1.

(A) The stability of reporters during in vitro translation. Shown are mRNA northern blots monitoring reporter mRNA levels over the course of the indicated in vitro translation reactions. (B) Schematic of mRNA cleavage and probing strategy used in RNase H northern blots, which were used to examine tail lengths. A DNA oligonucleotide complimentary to the 3′-UTR was used to direct cleavage of the target mRNA by RNase H, leaving a 40-nt fragment of the 3′-UTR appended to the poly(A) tail, which was resolved on a denaturing gel and detected by a radiolabeled probe. (C) The integrity of reporter poly(A) tails during in vitro translation. Shown are RNase H northern blots monitoring poly(A)-tail lengths of reporter mRNAs in in vitro translation reactions, as illustrated in (B). Reference tail lengths are inferred from lengths of size markers. (D) Purification of PABPC1. At the top is a chromatogram of cation exchange after nickel affinity columns. In the middle is a chromatogram of gel filtration after the cation exchange. At the bottom are Coomassie-stained SDS-PAGE gels monitoring fractions collected during the cation exchange (Mono S) and the gel filtration (Superdex 200). Green fractions were pooled after the cation-exchange step, and red fractions were pooled after the gel-filtration step. (E) Expression of proteins from mRNAs injected into frog oocytes. Shown is a western blot detecting proteins from frog oocytes in which mRNAs were injected for expressing the indicated proteins. (F) The integrity of reporter tail lengths after injection into oocytes. Shown is an RNase H northern blot monitoring poly(A)-tail lengths of reporter mRNAs injected into oocytes. Otherwise this panel is as in (C).
Figure 1—figure supplement 2. Additional reporter assays examining the effect of PAPBC levels on coupling between tail length and translation.

Figure 1—figure supplement 2.

(A) The effect of tail length on reporter translation in vitro. This panel is as in Figure 1B, but uses Rluc reporters instead of Nluc reporters and shows the results of one replicate. (B) The effect of PABPC1 or ePAB overexpression on relative translation of short- and long-tailed reporter mRNAs in frog oocytes. This panel is as in Figure 1E but uses Rluc reporters instead of Nluc reporters. (C) The effect of PABPC1 and eIF4G overexpression on translation of Nluc reporter mRNAs in frog oocytes. Expression of different proteins was achieved by injecting the indicated amount of mRNA in the first injection. Otherwise, this panel is as in (B).