Dynamic U2AF cycling defines two phases of cotranscriptional pre-mRNA splicing.

Distinguishing functional splice sites from abundant cryptic sites in precursor messenger RNAs (pre-mRNAs) represents a fundamental challenge in decoding mammalian genomes. We demonstrate that the specific RNA polymerase II (Pol II) subunit RPB9 directly interacts with the 3′ AG dinucleotide binding factor U2AF1 to initiate 3′ splice site recognition. Combined with recent structural insights into Pol II–mediated 5′ splice site selection, these findings support a cotranscriptional mechanism to recognize paired 3′ and 5′ splice sites across individual exons. These initial exon definition events facilitate the recruitment of U2AF2 to heterodimerize with U2AF1, which also triggers U2AF1 release from elongating Pol II. Collectively, these results reveal dynamic U2AF cycling that partitions Pol II subunit–facilitated splice site recognition and subsequent Pol II–independent spliceosome assembly steps during cotranscriptional splicing. Editor's summary: In eukaryotic cells, transcription and RNA splicing are tightly linked, but how these processes are coordinated remains unclear. Shao et al. found that the RNA polymerase II (Pol II) C-terminal domain was not required for recruiting splicing factors during co-transcriptional splicing. Instead, the Pol II subunit RPB9 directly interacted with the splicing factor U2AF1, whereas U2AF2 operated independently. There is thus a dynamic "U2AF cycle" that separates splicing into two phases: an early Pol II–dependent phase and a later Pol II–independent phase. These findings reshape our understanding of how splicing is regulated during transcription, with implications for gene expression and disease mechanisms. —Di Jiang INTRODUCTION: In eukaryotic cells, transcription is coupled with precursor messenger RNA (pre-mRNA) splicing to enable linear presentation of functional splice sites (ss) to the splicing machinery. This mechanism allows introns to be removed before RNA polymerase II (Pol II) completes transcription. Mechanistically, cotranscriptional splicing may occur in two phases: (i) a Pol II–mediated phase, evidenced by Pol II–associated splicing factors, and (ii) a Pol II–independent phase operating on nascent RNA attached to elongating Pol II, potentially influenced by and/or contributing to the local chromatin environment. It has remained unclear whether these two phases are differentially partitioned among introns and how such sequential events are dynamically regulated. RATIONALE: The C-terminal domain (CTD) of Pol II's largest subunit, RPB1, has long been hypothesized to facilitate cotranscriptional splicing, but the requirement of the CTD for recruiting Pol II–associated splicing factors has yet to be directly tested. Conversely, increasing evidence points to CTD-independent mechanisms for cotranscriptional splicing, as exemplified by the recently visualized interaction between U1 small nuclear ribonucleoprotein (snRNP) and Pol II subunits RPB2 and RPB12. This study began with the identification of another Pol II subunit, RPB9, in regulated splicing, leading to the elucidation of an unprecedented U2 auxiliary factor (U2AF) cycle that partitions the two phases of cotranscriptional splicing. RESULTS: We first directly tested whether the Pol II CTD is required to recruit several well-established Pol II–associated splicing factors and found that truncation of the CTD did not affect their association with Pol II. In contrast, we demonstrated that RPB9 mediates Pol II to directly interact with U2AF1, the small subunit of the U2AF heterodimer responsible for defining functional 3′ss. Unexpectedly, U2AF2, the large subunit of the U2AF heterodimer, was not part of the Pol II complex. We further showed that monomeric U2AF2 can release U2AF1 from immobilized RPB9 in a concentration-dependent manner. Given that U2AF is perceived as an inseparable heterodimer (stable even in 2 M guanidine chloride), the newly identified monomeric functions of U2AF1 and U2AF2 suggest a U2AF cycle that segregates the two phases of cotranscriptional splicing. This dynamic U2AF cycle aligns with distinct genome-wide association profiles: U2AF1 primarily tracks with Pol II on DNA, while U2AF2 predominantly binds pyrimidine-rich RNA. Analysis of chromatin-associated RNA revealed synergistic functions of RPB9 and U2AF1 in cotranscriptional splicing, whereas CTD truncation had only a modest impact. CONCLUSION: Cotranscriptional splicing involves early Pol II–mediated splice site recognition events, followed by the release of the initial exon definition complex to complete subsequent spliceosome assembly steps on nascent RNA. This segregation of Pol II–dependent and Pol II–independent phases is orchestrated by the cotranscriptional formation of the U2AF heterodimer. Certain late spliceosome steps and the splicing reaction itself may take place either before or after transcription completion, with different introns dictating differential splicing kinetics. Two phases of cotranscriptional splicing regulated by the U2AF cycle.: During transcription elongation, U2AF1 travels with Pol II to facilitate 3′ss recognition. This process likely coordinates with simultaneous SR protein binding of exonic splicing enhancers and Pol II–facilitated U1 snRNP recognition of the downstream 5′ss. This initial exon definition complex is released from Pol II upon U2AF2 recruitment, which may simultaneously engage in interactions with U2AF1, exon-bound SR proteins, and the newly emerged polypyrimidine tract as part of the 3′ss. The released complex then undergoes further assembly on nascent RNA to form a catalysis-competent spliceosome. BPS, branchpoint sequence. [ABSTRACT FROM AUTHOR]