Fig 2: 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.

Fig 3: Colocalization images of ATXN2 in cytoplasmic organelle and related molecules in normal control brain. The confocal microscopic images of cytoplasmic ATXN2 and several organelle marker proteins, including endoplasmic reticulum marker Calnexin, ribosomal protein S6 (RPS6), Golgi Glycoprotein 1 (GLG1), Lysosome associated membrane protein 1 (LAMP1), ATXN2’s related protein poly-A binding protein 1 (PABP1) and TDP-43, are shown. a–f Representative fluorescent double-stained images from the upper layer of the cerebral temporal cortex, including merged images with DAPI staining and two-dimensional scatterplots. ATXN2 is partially localized within the endoplasmic reticulum (a) and is strongly associated with ribosomes (b) and PABP1 (e) in controls, while ATXN2 did not localize within the Golgi apparatus (c) and lysosomes (d). There was no colocalization between ATXN2 and TDP-43 (f). Pseudo coloring was used for each channel

Fig 4: MKRN1 is a sensor for poly(A) sequences that stalls ribosomes to initiate ribosome-associated quality control. Proposed model of MKRN1 function: MKRN1 is positioned upstream of (premature) poly(A) tails via interaction with PABPC1. Ribosomes translating the open reading frame run into MKRN1 that acts as a roadblock to prohibit poly(A) translation. Upon contact with the translating ribosome, MKRN1 ubiquitylates the 40S ribosomal protein RPS10. This stalls the ribosome, causing the trailing ribosomes to collide. ZNF598 recognizes the collided ribosomes and ubiquitylates ribosomal proteins to promote RQC

Fig 5: T6B fusion protein prevents miRNA-induced silencing complex (miRISC) assembly and impairs microRNA (miRNA) activity in vitro.(A) Schematics of T6B action: T6B competes with TNRC6 for binding to AGO proteins preventing miRISC assembly. (B) Schematics of the size-exclusion chromatography (SEC) assay for the fractionation of AGO-containing complexes according to their molecular weight. (C) SEC profiling of miRISC components upon T6B expression: total lysates from HCT116 cells expressing no fusion protein (upper panel), T6B (middle panel), or T6BMut (lower panel) were fractionated as described in (B) and immunoblotted to detect AGO2, TNRC6A, T6B, and PABP1. For each blot, the relative signal intensity was assessed by densitometric analysis. (D) RNAseq analysis of total and small RNAs isolated from mouse embryo fibroblasts (MEFs) cell lines expressing either no fusion protein, T6B, or T6BMut (n = 3 for each cell line). Upper panel: bubble plot of target de-repression against miRNA abundance. The mean log2-fold change (T6B or T6BMut vs. control) of predicted targets for each conserved miRNA family was calculated, converted to a z-score and is plotted on the x-axis against the miRNA family abundance (log of the sum of read counts for each member of the family). The size of each circle is proportional to the number of predicted targets. A positive z-score indicates that the targets for that family are preferentially upregulated upon T6B expression, while a negative score would indicate preferential downregulation. Expression of T6B, but not of T6BMut, causes preferential upregulation of miRNA targets of the most miRNA families and the effect is roughly proportional to each miRNA family abundance. Lower panel: cumulative distribution plot of predicted let-7 targets compared to background in T6B-expressing MEFs. (E) Scatter plots of miRNA abundance as determined by small-RNAseq of total RNA extracted from MEFs expressing either T6B or T6BMut (n = 3 for each cell line). Each dot represents a miRNA in miRbase. (F) Effect of T6B expression on AGO2 slicing activity. MEFs expressing either T6B or T6BMut were transfected with siRNAs targeting GAPDH mRNA (siGAPDH) or with scramble siRNA (siCTL). Levels of GAPDH, T6B, and tubulin were assessed by immunoblot 72 hr post-transfection. T6B and T6BMut have slightly different migration on PAGE, as previously observed by Hauptmann et al., 2015. Figure 1—source data 1.RNAseq, differential gene expression, mouse embryo fibroblasts. Figure 1—source data 2.z-scores and miRNA family abundance, mouse embryo fibroblasts. Figure 1—source data 3.Small RNAseq, microRNA counts, mouse embryo fibroblasts. Figure 1—source data 4.Unedited blots shown in Figure 1C. Figure 1—source data 5.Unedited blots shown in Figure 1F. Figure 1—source data 6.Unedited blots shown in Figure 1F. Figure 1—source data 7.Uncropped blots shown in Figure 1F.