SummaryWe present a molecular dissection of pause site-dependent transcriptional termination for mammalian RNA polymerase II (Pol II)-transcribed genes. We show that nascent transcripts form RNA/DNA hybrid structures (R-loops) behind elongating Pol II and are especially prevalent over G-rich pause sites positioned downstream of gene poly(A) signals. Senataxin, a helicase protein associated with AOA2/ALS4 neurodegenerative disorders, acts to resolve these R-loop structures and by so doing allows access of the 5′–3′ exonuclease Xrn2 at 3′ cleavage poly(A) sites. This affords 3′ transcript degradation and consequent Pol II termination. In effect, R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are key steps in the termination process.

transferase activities are part of the same polypeptide University of Oxford at the N terminus and C terminus, respectively, while South Parks Road in yeast they are catalyzed by separate enzymes. This Oxford OX1 3RE initial cap structure is then recognized by the cap bind-United Kingdom ing complex (CBC), which contains two proteins, CBP20 and CBP80. Upon export through the nuclear pore complex, the nuclear cap binding proteins are replaced by The messenger RNA processing reactions of capping, the cytoplasmic translation initiation factor, eIF-4E splicing, and polyadenylation occur cotranscription-(Shatkin and Manley, 2000, for review). ally. They not only influence one another's efficiency The cap structure bound to CBC is believed to play and specificity, but are also coordinated by transcripa major role in the stabilization of the mRNA, since it tion. The phosphorylated CTD of RNA polymerase II represents an obstacle for 5Ј-3Ј exonucleases (Beelman provides key molecular contacts with these mRNA and Parker, 1995). In addition, in the cytoplasm, cap processing reactions throughout transcriptional elonbound to eIF-4E and other translation initiation factors gation and termination.enhances translation by promoting the engagement of the ribosomal subunits with the mRNA. This is at least partly achieved by the interaction of eIF4G with poly(A)-

The pathway from gene activation in the nucleus to mRNA translation and decay at specific locations in the cytoplasm is both streamlined and highly interconnected. This review discusses how pre-mRNA processing, including 5' cap addition, splicing, and polyadenylation, contributes to both the efficiency and fidelity of gene expression. The connections of pre-mRNA processing to upstream events in transcription and downstream events, including translation and mRNA decay, are elaborate, extensive, and remarkably interwoven.

SummaryTranscription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5′splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

Polyadenylation [poly(A)] signals (PAS) are a defining feature of eukaryotic protein-coding genes. The central sequence motif AAUAAA was identified in the mid1970s and subsequently shown to require flanking, auxiliary elements for both 39-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination. More recent genomic analysis has established the generality of the PAS for eukaryotic mRNA. Evidence for the mechanism of mRNA 39-end formation is outlined, as is the way this RNA processing reaction communicates with RNA polymerase II to terminate transcription. The widespread phenomenon of alternative poly(A) site usage and how this interrelates with pre-mRNA splicing is then reviewed. This shows that gene expression can be drastically affected by how the message is ended. A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.