Global insights into alternative polyadenylation regulation

Abstract

Alternative pre-mRNA processing greatly increases the coding capacity of the human genome and regulatory factors involved in RNA processing play critical roles in tissue development and maintenance. Indeed, abnormal functions of RNA processing factors have been associated with a wide range of human diseases from cancer to neurodegenerative disorders. While many studies have emphasized the importance of alternative splicing (AS), recent high-throughput sequencing efforts have also allowed global surveys of alternative polyadenylation (APA). For the majority of pre-mRNAs, as well as some non-coding transcripts such as lncRNAs, APA selects different 3'-ends and thus modulates the availability of regulatory sites recognized by trans-acting regulatory effectors, including miRs and RNA binding proteins (RBPs). Here, we compare the available technologies for assessing global polyadenylation patterns, summarize the roles of auxiliary factors on APA, and discuss the impact of differential polyA site (pA) selection in the determination of cell fate, transformation and disease.

Keywords: 3′ UTR, 3′-untranslated region; APA, Alternative polyadenylation; AS, Alternative splicing; DM, Myotonic dystrophy; HITS-CLIP, High-throughput sequencing coupled with crosslinking and immunoprecipitation; KD, Knockdown; KO, Knockout; MBNL; PolyA-seq; RBP, RNA binding protein; RNA processing; alternative polyadenylation; microsatellites; myotonic dystrophy; neurological disease; pA, Polyadenylation site.

Figure 1.

Alternative Polyadenylation Site Identification by RNA-seq versus PolyA-seq. Comparison of PolyA-seq and RNA-seq wiggle plots of Bicd2 (left), Capzb (middle), and Rbm39 (right) in WT (blue) and Mbnl1;Mbnl2 DKO MEFs (red). RNA-seq detects internal 3′ UTR splicing in Bicd2 but fails to capture the pA shift in Capzb. Both techniques detect the pA shift in Rbm39.

Figure 2.

Mechanisms of RBP-mediated APA regulation. This illustration highlights the 3′ UTR (gray bar) downstream of the stop codon (red octagon) and the cis elements that bind the core 3′-end processing machinery. While the proximal site (pA1) is weaker (AUUAAA), the distal site (pA2) contains the canonical AAUAAA hexamer. Also shown are the U-rich upstream sequence element (USE) recognized by cleavage factor I_m (CFI_m), the U/GU-rich downstream sequence element (DSE) that binds cleavage stimulation factor (CstF) and the polyA signal (PAS) A(A/U)UAAA recognized by the cleavage and polyadenylation specificity factor (CPSF). Cleavage occurs 3′ of the CA dinucleotide (red). Also included in the figure are several examples of 3′-end processing modulated by RBP 3′ UTR binding: 1) In the nucleus, the cytoplasmic polyadenylation element binding protein 1 (CPEB1) recognizes the cytoplasmic polyadenylation element (CPE) upstream of the weaker proximal pA1 and recruits the CPSF complex to promote 3′ UTR shortening; 2) hnRNP H binds in the proximity of pA1 to recruit the core processing machinery to cause 3′ UTR shortening; 3) NOVA2 binds near pA1 to suppress proximal usage; 4) PolyA binding protein nuclear 1 (PABPN1) recognizes PASs and binds to the weaker pA1 thereby blocking CPSF binding (in OPMD, insufficient proximal pA1 suppression by PABPN1 leads to 3′ UTR shortening and PABPN1 cannot compete with CPSF for the strong canonical PAS); 5) MBNL proteins act by blocking the recruitment of the core machinery to intronic (not shown), proximal, and sometimes distal (not shown) sites when they bind within ± 50–100 nt of the pA site; 6) MBNL proteins also enhance pA selection by recruiting core polyadenylation factors.