Mechanisms and consequences of alternative polyadenylation

Abstract

Alternative polyadenylation (APA) is emerging as a widespread mechanism used to control gene expression. Like alternative splicing, usage of alternative poly(A) sites allows a single gene to encode multiple mRNA transcripts. In some cases, this changes the mRNA coding potential; in other cases, the code remains unchanged but the 3' UTR length is altered, influencing the fate of mRNAs in several ways, for example, by altering the availability of RNA binding protein sites and microRNA binding sites. The mechanisms governing both global and gene-specific APA are only starting to be deciphered. Here we review what is known about these mechanisms and the functional consequences of alternative polyadenylation.

Figure 1. Schematic representation of CR-APA and UTR-APA

CR-APA produces mRNA isoforms with distinct C-terminal coding regions, resulting in distinct protein isoforms. UTR-APA produces distinct mRNA isoforms with different length 3’UTRs, but encode the same protein. Longer 3’UTRs usually contain cis-regulatory elements, such as miRNA and/or protein binding sites, which often bring about mRNA instability or translational repression. CR-APA, coding region-alternative polyadenylation; UTR-APA, 3’UTR-alternative polyadenylation. Light green boxes, untranslated regions; blue light blue boxes, shared coding regions; dark blue and yellow boxes, unshared coding regions; lines, introns.

Figure 2. Connecting APA to cellular proliferative and developmental states

Enhanced proliferation such as during de-differentiation (e.g., in the generation of iPS cells), T cell activation, or cellular transformation are associated with upregulation in expression of certain 3’ processing factors and with increased usage of proximal poly(A) sites. Late developmental stages and cellular differentiation (e.g. differentiation of C2C12 into myotubes) are associated with downregulation of expression of 3’ processing factors and increased usage of distal poly(A) sites.

Figure 3. Examples of gene regulation by APA

(A) The immunoglobulin heavy chain M gene is partly shown; a constant regions (Cμ4) is shared by both µm and µs mRNAs, while exons M1 and 2 (yellow boxes) and S (red boxes) are specific to µm and µs mRNAs, respectively. In resting B cells, the amount of CstF is limiting, and the distal poly(A) site, which binds CstF more avidly, is preferentially used, resulting in production of the membrane-bound form of IgM (µm). In activated B cells, the concentration of CstF is elevated and no longer limiting, so the proximal, first transcribed poly(A) site is preferentially selected, leading to production of secreted-form IgM (µs). Additional factors, such as the transcription factor Ell2 (see text), may also contribute to the switch. (B) CyclinD1 is subject to both UTR-APA and CR-APA. Two major isoforms are created by CR-APA: cyclin D1a (full-length isoform) and cyclin D1b (truncated isoform). The truncated isoform is associated with a polymorphism at the end of exon 4 (E4) (G870A, arrowhead), resulting in increased usage of the poly(A) site located in intron 4 (I4). This isoform is retained in the nucleus and is associated with increasing transforming capability. UTR-APA of CyclinD1 leads to increased usage of the weak proximal poly(A) site in cancer cells generally, or in the usage of a newly mutational-derived proximal poly(A) site in Mantle Cell Lymphoma. In both, mRNAs with shorter 3’UTRs are generated. Light green boxes, untranslated regions; blue light blue boxes, shared coding regions; lines, introns. (C) Seasonal flowering control by antisense-RNA transcript. FPA and FCA promote selection of the proximal poly(A) site of an antisense transcript that initiates downstream of the FLC gene (red arrows) by stimulating 3’ end formation at that site. 3’ end processing at the proximal poly(A) site recruits the histone demethylase, FLD, which induces histone modifications on internal nucleosomes that result in silencing the sense FLC transcript (blue arrow). In the absence of FPA and FCA, the distal poly(A) site of the antisense transcripts is selected. This may facilitate the recruitment of positive transcription factors to the FLC promoter, resulting in activation of FLC transcription.

Figure 4. Mechanisms regulating APA

The choice of using one poly(A) site over another is dictated by a combination of several features. (A) Variations in the abundance or activity of trans-acting factors such as core 3’ processing proteins and tissue-specific RNA-binding proteins, as well as through interaction with splicing and transcription factors. (B) Combinations of cis-acting RNA elements, such as the strength of binding sites for core 3’ processing factors, auxiliary sequences and/or new motifs directing the interaction of protein components with the mRNA, and perhaps RNA secondary structures. (C) APA is likely also influenced by chromatin, including nucleosome positioning around the poly(A) site, DNA methylation and histone post-translational modifications.