An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries.

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing

Human 5' --> 3' exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites.

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

p54(nrb) associates with the 5′ splice site within large transcription/splicing complexes

The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54(nrb), a transcription/splicing factor, associates with the 5′ splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO–snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO–snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54(nrb) and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5′ SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process