mRNA Polyadenylation

mRNA Polyadenylation All eukaryotic mRNAs, with the exception of histone mRNAs, undergo a 3' end maturation step consisting of a specific endonucleolytic cleavage of the precursor followed by polyadenylation of the upstream cleavage fragment; the downstream fragment is degraded (reviewed in Gruber et al. 2014, Eaton and West 2020). In mammalian cells, the pre-mRNA cleavage site is determined by at least four sequence elements (reviewed in Gruber and Zavolan 2019). 1) The central and most highly conserved signal is AAUAAA or a close variant located ~20 nucleotides (nt) upstream of the cleavage site. 2) The preferred sequence at the cleavage site is CA. 3) GU- or G-rich downstream elements are important, and 4) sequences upstream of AAUAAA, such as UGUA, can also contribute. The majority of protein-coding genes have multiple polyadenylation sites (PASs) generating either different protein isoforms or mRNA isoforms differing in the lengths of their 3' untranslated regions (UTRs) and consequently in their interaction with RNA-binding proteins and microRNAs (reviewed in Gruber et al. 2014, Tian and Manley 2014, Gruber and Zavolan 2019). Formation of mRNA isoforms through differential usage of PASs is called alternative polyadenylation (APA) (reviewed in Xu et al. 2022). APA is frequently used in all eukaryotes, and more than 70% of protein-coding mRNAs in mammals are subject to APA (reviewed in Guo and Lin 2021).In mammalian cells, more than twenty core proteins organized into several different complexes are dedicated to the cleavage and polyadenylation (CP) reaction (reviewed in Gruber and Zavolan 2019). A much larger set of ~80 proteins has been identified by affinity purification of a mammalian 3' processing complex and mass spectrometric analysis (Shi et al. 2009; reviewed in Gruber and Zavolan 2019). Some of these proteins may contribute to the coupling of 3' processing to transcription and other processes (Gruber et al. 2014). The central complex is the cleavage and polyadenylation specificity factor (CPSF). CPSF carries the catalytic activity for pre-mRNA cleavage, and its interaction with the AAUAAA sequence is essential for cleavage and the AAUAAA dependence of polyadenylation (reviewed in Gruber et al. 2014, Tian and Manley 2014, Gruber and Zavolan 2019). The CPSF complex is thought to consist of seven proteins: CPSF1 (CPSF160), CPSF2 (CPSF100), CPSF3 (CPSF73), CPSF4 (CPSF30), FIP1L1 (FIP1), WDR33, and SYMPK (Symplekin) (reviewed in Gruber et al. 2014, Tian and Manley 2014). SYMPK is considered to be a scaffolding factor, as it interacts with both the CPSF complex and the CSTF complex (reviewed in Tian and Manley 2014). The cleavage stimulation factor (CSTF) complex has three different subunits, CSTF1, CSTF2 and CSTF3, and recognizes downstream elements (Takagaki and Manley 2000, reviewed in Gruber and Zavolan 2019). The cleavage factor I (CF I) complex recognizes the UGUA upstream element (reviewed in Gruber and Zavolan 2019) . The CF I complex is a heterotetramer consisting of a homodimer of NUDT21 (CPSF5) and a homo- or a heterodimer of CPSF6 and CPSF7 (Kim et al. 2010). The cleavage factor II (CF II) complex is a heterodimer of two subunits, CLP1 and PCF11. The function of CF II is poorly studied. It has been proposed that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements (Schäfer et al. 2018, reviewed in Gruber and Zavolan 2019). The poly(A) polymerase generates the poly(A) tail and can also contribute to cleavage (reviewed in Gruber and Zavolan 2019). Although CPSF and poly(A) polymerase are sufficient for AAUAAA-dependent polyadenylation, the nuclear poly(A)-binding protein 1 (PABPN1) stimulates poly(A) tail extension and is essential for the synthesis of a poly(A) tail of the appropriate length (reviewed in Gruber and Zavolan 2019). Three related RNA poly(A) polymerases exist in mammals, PAPOLA, PAPOLB and PAPOLG. PAPOLA, also known as PAP or poly(A) polymerase alpha or PAPII, is considered to be the canonical poly(A) polymerase in mRNA polyadenylation. PAPOLG, commonly known as poly(A) polymerase gamma, localizes to the nucleus like PAPOLA, and co-immunoprecipitates with the 3' processing complex (Shi et al. 2009). PAPOLG shares 60% identity to human PAPOLA at the amino acid level. PAPOLG exhibits fundamental properties of a bona fide poly(A) polymerase, specificity for ATP, and CPSF/AAUAAA-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of PAPOLA. PAPOLG and PAPOLA have similar organization of structural and functional domains. PAPOLG contains a U1A protein-interacting region in its C terminus, and PAPOLG activity can be inhibited, as PAPOLA, by the U1A protein (Kyriakopoulou et al. 2001). PAPOLG may also function in monoadenylation of small RNAs (Perumal et al. 2001). PAPOLB, also known as TPAP or poly(A) polymerase beta, is a cytosolic poly(A) polymerase specifically expressed in the testis. PAPOLB is thought to govern germ cell morphogenesis by modulating specific transcription factors at posttranscriptional and posttranslational levels (Kashiwabara et al. 2000; Kashiwabara et al. 2002; Kashiwabara et al. 2016). An additional nuclear poly(A) polymerase in mammals is TUT1 (also known as STAR-PAP or STPAP), which preferentially polyadenylates pre-mRNAs of oxidative stress-induced genes (Mellman et al. 2008; Li et al. 2012; reviewed in Li et al. 2013).