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.