SummarySen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
SummaryTranscription termination by RNA polymerase II is coupled to transcript 3′ end formation. A large cleavage and polyadenylation complex containing the major poly(A) polymerase Pap1 produces mRNA 3′ ends, whereas those of nonpolyadenylated snoRNAs in yeast are formed either by endonucleolytic cleavage or by termination, followed by trimming by the nuclear exosome. We show that synthesis of independently transcribed snoRNAs involves default polyadenylation of two classes of precursors derived from termination at a main Nrd1/Nab3-dependent site or a “fail-safe” mRNA-like signal. Poly(A) tails are added by Pap1 to both forms, whereas the alternative poly(A) polymerase Tfr4 adenylates major precursors and processing intermediates to facilitate further polyadenylation by Pap1 and maturation by the exosome/Rrp6. A more important role of Trf4/TRAMP, however, is to enhance Nrd1 association with snoRNA genes. We propose a model in which polyadenylation of pre-snoRNAs is a key event linking their transcription termination, 3′ end processing, and degradation.
Transcribing RNA Polymerase II interacts with multiple factors that orchestrate maturation and stabilisation of messenger RNA. For the majority of noncoding RNAs, the polymerase complex employs entirely different strategies, which usually direct the nascent transcript to ribonucleolytic degradation. However, some noncoding RNA classes use endo-and exonucleases to achieve functionality. Here we review processing of small nucleolar RNAs that are transcribed by RNA Polymerase II as precursors, and whose 5 0 and 3 0 ends undergo processing to release mature, functional molecules. The maturation strategies of these noncoding RNAs in various organisms follow a similar pattern but employ different factors and are strictly correlated with genomic organisation of their genes. Small Nucleolar RNAs: Essential and Functional Noncoding RNAsDuring the past decade, it has become apparent that RNAs transcribed from noncoding regions of the genome play essential functions in various biological processes. This prompted researchers to gain an understanding of how noncoding RNA (ncRNA) biogenesis is controlled and whether (and how) this process resembles synthesis of messenger RNA (mRNA). Recent analyses of small nucleolar RNAs (snoRNAs) have provided fundamental information on how expression of these ncRNAs is regulated. SnoRNAs are essential, short (60-300 nt long), mostly nucleoli-localised, non-polyadenylated ncRNAs, present in all eukaryotic organisms. They are classified by the presence of highly conserved sequences ('boxes'), as either box C/D or box H/ACA (Figure 1A). The box C/D snoRNAs form a closed loop, which contains a box C and a box D (with conserved RUGAUGA and CUGA motifs, respectively), as well as a less conserved box C 0 and box D 0 [1]. The box H/ACA snoRNAs usually consist of two stem loops linked by the H box (ANANNA motif) and an ACA sequence near the 3 0 end [1]. Box C/D snoRNAs with a long UG repeat and H/ACA snoRNAs with an additional CAB box (UGAG motif) are called small Cajal body-associated RNAs (scaRNA) (Figure 1A) and are localised in subnuclear structures known as Cajal bodies [2,3]. Both snoRNA classes are bound by a distinct set of proteins to form stable and catalytically active box C/D and box H/ACA ribonucleoprotein (snoRNP) structures [4].
In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo.
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