The 5' fragment generated by 3' cleavage of the pre-mRNA ...

The 5' fragment generated by 3' cleavage of the pre-mRNA receives a poly(A) tail of approximately 250 AMP residues in a reaction depending on the AAUAAA polyadenylation sequence (PAS) 10 to 30 nucleotides upstream of the 3' cleavage site and catalyzed by one of three nuclear RNA polyA polymerases (PAPs), PAPOLA (also known as PAPalpha), PAPOLG (also known as PAPgamma, or TUT1 (also known as STAR-PAP) (Li et al. 2017). Functions of PAPOLA, PAPOLG and TUT1 are partially redundant, but each PAP is also specifically involved in polyadenylation of a subset of transcripts (Li et al. 2017). PAPOLG structure closely resembles the structure of PAPOLA (Yang et al. 2014). TUT1 activity is regulated by nuclear phosphoinositide signaling, specifically nuclear phosphatidylinositol-4,5-bisphosphate, PI4,5P(2), and TUT1 specifically promotes polyadenylation and expression of mRNAs produced by genes involved in oxidative stress response, such as HMOX1 (Mellman et al. 2008; Li et al. 2012; reviewed in Li et al. 2013), and, in general, genes with low expression levels (Li et al. 2017).

The activity of PAPs depends on two auxiliary factors, both of which bind to specific RNA sequences and recruit the enzyme by a direct contact. One of these factors is the CPSF complex, which binds the AAUAAA sequence and is also essential for 3' cleavage (Shi et al. 2009, Zhang et al. 2020). FIP1L1 (also known as FIP1), the subunit of the CPSF submodule mPSF, interacts with the structured core of human PAPOLA, with a binding mode that is evolutionarily conserved from yeast to human (Todesca et al. 2024). In higher eukaryotes, however, a conserved C-terminal motif of PAPOLA intramolecularly interacts with the FIP1L1-binding region of PAPOLA (Todesca et al. 2024). Interaction of the C-terminal motif of PAPOLA with another CPSF subunit, CPSF1 (also known as CPSF160) is required to enable PAPOLA interaction with FIP1L1 (Todesca et al. 2024). The second factor is the nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once they reach a length of about ten nucleotides, increasing PAP processivity to enable synthesis of full-length poly(A) tails of ~250 nt (Wahle et al. 1991: bovine PABPN1 was used; Wahle et al. 1995: bovine PABPN1 was used; Kerwitz et al. 2003: bovine PABPN1 was used; Kühn et al. 2017: human PABPN1 was used).

Strong distal 3'UTR PASs may be processed first, with the resulting longer transcripts subsequently anchored in chromatin/nuclear matrix to serve as precursors for further processing at proximal 3'UTR PASs, implying that alternative polyadenylation (APA) sites are recognized independently, and in many cases, in a sequential manner (Tang et al. 2022).