RNA polymerase II (RNA Pol II) slows around the polyA sig...

RNA polymerase II (RNA Pol II) slows around the polyA signal site (PAS), a hexameric AAUAAA sequence in the pre-mRNA (Tran et al. 2001), and shows increased Ser2 phosphorylation of the C-terminal domain (CTD) by Ser2 kinases such as CDK12, which promotes recruitment of 3'-end processing factors (Davidson et al. 2014). Some components of the core cleavage and polyadenylation (CPA) complex may be recruited to the RNA Pol II during transcription initiation (Davidson et al. 2014), but the available information is insufficient to show this in the diagram. Instead, a simplified, linear assembly of the CPA complex is depicted, with target pre-mRNA bound to the early spliceosome (Spliceosomal A complex).

The cleavage factor I (CF I, also known as CFIm) subcomplex of the core CPA complex recognizes the PAS upstream element UGUA, with the NUDT21 subunit of the CF I complex binding the UGUA element in a sequence-specific manner (Yang et al. 2011). The cleavage and polyadenylation specificity factor (CPSF) subcomplex of the core CPA complex directly recognizes the PAS, with its subunits WDR33 and CPSF4 (also known as CPSF30) binding specifically to the AAUAAA hexamer (Chan et al. 2014; Schönemann et al. 2014; Clerici et al. 2017; Sun et al. 2018; Hamilton et al. 2019). The CPSF1 (also known as CPSF160) subunit of the CPSF subcomplex functions as a scaffold that organizes CPSF4 and WDR33 to ensure high-affinity binding to the PAS (Clerici et al. 2017; Sun et al. 2018; Hamilton et al. 2019), and the FIP1L1 (also known as FIP1) subunit also aids the PAS recognition (Clerici et al. 2017). The CSTF2 (also known as CSTF64) subunit of the CSTF subcomplex binds to the pre-mRNA U/GU-rich elements downstream of the PAS site via its RNA recognition motif (RRM) (MacDonald et al. 1994; Masoumzadeh and Latham 2024). The CSTF3 (also known as CSTF77) subunit of the CSTF complex stimulates the RNA binding of CSTF2 (Grozdanov et al. 2018), and CSTF1 (also known as CSTF50) fine tunes the recognition of the target sequence by the RNA recognition motif (RRM) of CSTF2 (Yang et al. 2018). CSTF2 also interacts with SYMPK (Symplekin) (Takagaki and Manley 2000; Yao et al. 2013), a protein known to be closely associated with the CPSF complex (Shi et al. 2009; Schönemann et al. 2014).

The CPA assembly on pre-mRNA occurs while splicing is still ongoing; many transcripts undergo 3'-end processing before completion of terminal intron splicing (reviewed in Misra and Green 2016). The polyA tail facilitates splicing of 3' end introns with weak 3' splice sites, acting as a splicing enhancer together with the polyA-binding protein PABPN1 (Huang et al. 2023: human cells were used). In addition, components of splicing machinery play regulatory roles in polyadenylation. U1 snRNP were shown to transiently associate with sequences upstream of PASs, through RNA-RNA interactions between the U1 snRNA and pre-mRNA, and repress the usage of cryptic PASs, such as the intronic PASs near the 5' end of gene, a mechanism known as U1 snRNP telescripting (Shi et al. 2019: human cells were used; Li et al. 2015: mouse cells were used; reviewed in Ran et al. 2021). U2 snRNA suppresses the uses of PASs in introns that can be efficiently spliced out (Li et al. 2015). Early stages of spliceosome assembly (Sharma et al. 2014) are sufficient to couple polyadenylation with splicing, but the ongoing removal of introns is not critical (Davidson and West 2013). The interplay between transcription, splicing and polyadenylation is complex and not fully elucidated (reviewed in Misra and Green 2016, and Choquet et al. 2025). Splicing of single-intron pre-mRNAs and at least some internal introns of multi-intron pre-mRNAs requires prior polyadenylation (reviewed in Misra and Green 2016). While splicing of some introns does not require transcriptional pausing, other introns are retained if 3' end cleavage or pre-mRNA is inefficient (Reimer et al. 2021). Polyadenylation of multi-intron genes occurs before the splicing of the last exon and overall 40% of mammalian introns are spliced out after polyadenylation (reviewed in Choquet et al. 2025).