Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes

Abstract

SummaryPat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5′ to 3′ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3′ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

GC content shapes mRNA decay and storage in human cells

“…Unexpectedly, the correlation was however positive with translational derepression after siDDX6 (Spearman r = 0.45, p<0.0001; Figure S6E), indicating that PAT1B preferentially targets mRNAs that are translationally repressed by DDX6. Accordingly, these transcripts are prone to PB storage (Spearman r = 0.49, p<0.0001; Figure S6F), as reported previously [9]. Indeed, in contrast to DDX6 and XRN1 decay targets, PAT1B targets were strikingly AU-rich (Spearman r = -0.50, p < 0.0001; Figures 4B and S4A).…”

“…Group II list mRNAs, except TOP mRNAs, ATXN2 and 4E-T targets, had the exact mirror fate compared to group I lists: they were stabilized following PAT1B silencing ( Figure S7D), as previously reported for ARE-containing mRNAs and the targets of the ARE-BPs HuR and TTP [9], but not following DDX6 or XRN1 silencing ( Figure S7B,C); they were enriched in PBs and translationally more active after DDX6 silencing ( Figure S8A,B), which is consistent with the reported presence of most of these regulatory proteins in PBs [2,34].…”

“…PAT1B is a well-characterized direct DDX6 partner known for its involvement in mRNA decay [9,[26][27][28]. However, using our previous PAT1B silencing experiment in HEK293 cells [9], we surprisingly found a negative correlation between mRNA stabilization after PAT1B and after DDX6 silencing (Spearman r = -0.31, p<0.0001; Figure S6D; Table S1, sheet6), suggesting that they largely target separate sets of mRNAs. Unexpectedly, the correlation was however positive with translational derepression after siDDX6 (Spearman r = 0.45, p<0.0001; Figure S6E), indicating that PAT1B preferentially targets mRNAs that are translationally repressed by DDX6.…”

“…Altogether, we therefore favor the possibility that the mechanism of PAT1B-dependent decay is prominently 3' to 5' in human cells. It could involve the CCR4/CNOT deadenylase and the LMS1-7 complexes, as, despite their low abundance or small size, CNOT1 and LSM2/4 had high scores in our previous PAT1B interactome analysis [9,28].…”

“…The RBP PAT1B has also been defined as an enhancer of decapping, as it interacts with DDX6, the LSM1-7 heptamer ring and the decapping complex in mammalian cells [9], while in yeast Pat1p activates Dcp2 directly [10] and its deletion results in deadenylated but capped intact mRNA [11,12]. DDX6 and PAT1B interact with the CCR4-NOT deadenylase complex and the DDX6-CNOT1 interaction is required for miRNA silencing [9,[13][14][15]. DDX6 also binds the RBP 4E-T, another key factor in PB assembly, which in turn interacts with the capbinding factor eIF4E and inhibits translation initiation, including that of miRNA target mRNAs [16].…”