Eukaryotic protein-encoding genes possess poly(A) signals that define the end of the messenger RNA and mediate downstream transcriptional termination by RNA polymerase II (Pol II). Termination could occur through an 'anti-termination' mechanism whereby elongation factors dissociate when the poly(A) signal is encountered, producing termination-competent Pol II. An alternative 'torpedo' model postulated that poly(A) site cleavage provides an unprotected RNA 5' end that is degraded by 5' --> 3' exonuclease activities (torpedoes) and so induces dissociation of Pol II from the DNA template. This model has been questioned because unprocessed transcripts read all the way to the site of transcriptional termination before upstream polyadenylation. However, nascent transcripts located 1 kilobase downstream of the human beta-globin gene poly(A) signal are associated with a co-transcriptional cleavage (CoTC) activity that acts with the poly(A) signal to elicit efficient transcriptional termination. The CoTC sequence is an autocatalytic RNA structure that undergoes rapid self-cleavage. Here we show that CoTC acts as a precursor to termination by presenting a free RNA 5' end that is recognized by the human 5' --> 3' exonuclease Xrn2. Degradation of the downstream cleavage product by Xrn2 results in transcriptional termination, as envisaged in the torpedo model.

Polymerase II (Pol II) transcriptional termination depends on two independent genetic elements: poly(A) signals and downstream terminator sequences. The latter may either promote cotranscriptional RNA cleavage or pause elongating Pol II. We demonstrate that the previously characterized MAZ 4 pause element promotes Pol II termination downstream of a poly(A) signal, dependent on both the proximity of the pause site and poly(A) signal and the strength of the poly(A) signal. The 533 exonuclease Xrn2 facilitates this pausedependent termination by degrading the 3 product of poly(A) site cleavage. The human ␤-actin gene also possesses poly(A) site proximal pause sequences, which like MAZ 4 are G rich and promote transcriptional termination. Xrn2 depletion causes an increase in both steady-state RNA and Pol II levels downstream of the ␤-actin poly(A) site. Taken together, we provide new insights into the mechanism of pause site-mediated termination and establish a general role for the 533 exonuclease Xrn2 in Pol II termination.Transcriptional termination can be defined as cessation of RNA synthesis followed by polymerase-DNA dissociation. Correct termination serves to maintain an active cellular pool of RNA polymerase II (Pol II) and to insulate downstream promoters from elongating Pol II (11). Despite the fundamental importance of this process, its mechanism remains enigmatic. Transcripts of all protein-encoding genes, with the exception of histone genes, contain a poly(A) signal at their 3Ј end composed of an AAUAAA sequence positioned 10 to 50 nucleotides upstream of a GU-rich downstream sequence element (see references 20 and 38 for reviews). The 5Ј product of endonucleolytic cleavage between these two sequences is polyadenylated and therefore stabilized. The 3Ј product of the cleavage is unstable and rapidly degraded.A substantial body of evidence argues that cleavage and polyadenylation of Pol II transcripts occur cotranscriptionally, effectively releasing mRNA from the transcription site. Thus, defects in mRNA 3Ј-end processing cause the accumulation of pre-mRNA at nuclear transcription foci both in yeast (13) and in mammals (6). Furthermore, numerous studies have shown that the C-terminal domain of the Pol II large subunit promotes efficient cleavage and polyadenylation (see references 14, 18, and 21 for reviews). It is also well documented that for mRNAs with multiple poly(A) signals, selection of a downstream poly(A) site results in mRNA with internal unprocessed poly(A) signals (7, 10). If 3Ј processing occurred posttranscriptionally, then usage of upstream poly(A) signals would always predominate. Finally, the poly(A) site is necessary for efficient transcriptional termination. However, the site of Pol II release is often distinct from the gene's poly(A) site. Thus, Pol II terminates transcription at sites positioned between 100 bp and several kb downstream of the poly(A) site (2,8,10,12,29). These observations suggest a requirement for additional, distal sequences in the termination process.A sequence (G 5 ...

39 end formation of pre-mRNAs is coupled to their transcription via the C-terminal domain (CTD) of RNA polymerase II (Pol II). Nearly all protein-coding transcripts are matured by cleavage and polyadenylation (CPA), which is frequently misregulated in disease. Understanding how transcription is coordinated with CPA in human cells is therefore very important. We found that the CTD is heavily phosphorylated on Ser2 (Ser2p) at poly(A) (pA) signals coincident with recruitment of the CstF77 CPA factor. Depletion of the Ser2 kinase Cdk12 impairs Ser2p, CstF77 recruitment, and CPA, strongly suggesting that the processes are linked, as they are in budding yeast. Importantly, we additionally show that the high Ser2p signals at the 39 end depend on pA signal function. Down-regulation of CPA results in the loss of a 39 Ser2p peak, whereas a new peak is formed when CPA is induced de novo. Finally, high Ser2p signals are generated by Pol II pausing, which is a well-known feature of pA site recognition. Thus, a reciprocal relationship between early steps in pA site processing and Ser2p ensures efficient 39 end formation.

Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5 ′ → 3 ′ exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3 ′ flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3 ′ end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on proteincoding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.