System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA–protein complexes

Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA–protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)+ RNA–protein crosslinking data provide information on the organization of RNA–protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3′ end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well

Investigating the role of Dis2 phosphatase in RNA Polymerase II transcription termination in fission yeast

The end of the RNA polymerase II (Pol II) transcription cycle is a tightly regulated process that requires: (i) dissociation of elongation factors, (ii) recruitment of the 3' end processing machinery that is essential for premRNA cleavage and polyadenylation, and (iii) association of termination factors to dislodge Pol II from the DNA template. A characteristic feature of termination factors is that they directly interact with the phosphorylated C-terminal domain (CTD) of the largest subunit of Pol II (Rpb1). The CTD consists of heptad repeats (Y1S2P3T4S5P6S7) where Tyr1, Ser2, Ser5, Ser7 and Thr4 can be reversibly phosphorylated during the transcription cycle. The work presented here reveals that Thr4 phosphorylation levels are enriched at the 3' ends of protein-coding genes in fission yeast and identies Dis2 as a Pol II-CTD phosphatase that dephosphorylates Thr4 both in vitro and in vivo. Dis2 activity is required for Thr4 dephosphorylation at the polyadenylation site, for recruitment of termination factors Pcf11, Rhn1 and Seb1 and for normal Pol II termination. Interestingly, Seb1 cotranscriptional recruitment is shown to be dependent not on Dis2 activity on CTD but on its ability to dephosphorylate the elongation factor Spt5, suggesting that Dis2 regulates the elongation-to-termination transition by targeting multiple substrates.</p

Transcription and chromatin-based surveillance mechanism controls suppression of cryptic antisense transcription

Phosphorylation of the RNA polymerase II C-terminal domain Y(1)S(2)P(3)T(4)S(5)P(6)S(7) consensus sequence coordinates key events during transcription, and its deregulation leads to defects in transcription and RNA processing. Here, we report that the histone deacetylase activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of antisense transcription when RNA polymerase II phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, although single Hos2 and Set3 mutants have little effect, loss of Hos2 or Set3 combined with ssu72Δ results in a synergistic increase in antisense transcription globally and correlates with elevated sensitivity to genotoxic agents. We demonstrate a key role for the Ssu72/Hos2/Set3 mechanism in the suppression of cryptic antisense transcription at the 3′ end of convergent genes that are most susceptible to these defects, ensuring the fidelity of gene expression within dense genomes of simple eukaryotes

Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination

Summary: Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3′ end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain. : Timely and efficient transcription termination is essential for release of functional mRNAs as well as for Pol II recycling. Kecman et al. demonstrate that the conserved PP1 phosphatase Dis2 regulates transcription termination in fission yeast by mediating elongation to termination factor exchange and by dephosphorylating Pol II C-terminal domain. Keywords: RNA polymerase II, C-terminal domain, CTD, PP1 phosphatase, transcription termination, CTD phosphorylation, Spt5, CTD interacting domain, CI