Alternative polyadenylation in the nervous system: to what lengths will 3' UTR extensions take us?

Abstract

Alternative cleavage and polyadenylation (APA) can diversify coding and non-coding regions, but has particular impact on increasing 3' UTR diversity. Through the gain or loss of regulatory elements such as RNA binding protein and microRNA sites, APA can influence transcript stability, localization, and translational efficiency. Strikingly, the central nervous systems of invertebrate and vertebrate species express a broad range of transcript isoforms bearing extended 3' UTRs. The molecular mechanism that permits proximal 3' end bypass in neurons is mysterious, and only beginning to be elucidated. This landscape of neural 3' UTR extensions, many reaching unprecedented lengths, may help service the unique post-transcriptional regulatory needs of neurons. A combination of approaches, including transcriptome-wide profiling, genetic screening to identify APA factors, biochemical dissection of alternative 3' end formation, and manipulation of individual neural APA targets, will be necessary to gain fuller perspectives on the mechanism and biology of neural-specific 3' UTR lengthening.

Keywords: 3′ UTR; RNA-binding protein; alternative polyadenylation; miRNA; nervous system.

Figure 1. The cleavage and polyadenylation (CP) machinery at the polyA site

A multiprotein complex cleaves the mRNA 3′ terminus and adds an untemplated polyA tail. RNA polymerase II (Pol II) recruits polyA factors and enhances the cleavage reaction, and is thus considered part of the CP machinery. The cleavage and polyadenylation specificity factor (CPSF) complex recognizes the polyadenylation signal (PAS); CPSF160 binds the AAUAAA signal and CPSF executes endonucleolytic cleavage, preferably at CA dinucleotides. The Cleavage stimulation factor (CstF) complex aids CP by recognizing the downstream sequence element (DSE) via the CstF-64 subunit. Other numbers within complexes correspond to the names of additional factors. Other auxiliary polyA factors have been omitted for clarity, as have other physical connections that the CP machinery has with the transcription complex and splicing factors. USE, upstream sequence element; CFIm, Cleavage factor Im; PAP, polyA polymerase.

Figure 2. Experimental approaches to study alternative polyadenylation

The schematics at top left depict a model gene that expresses short (orange) and long (purple) 3′ UTR isoforms, of which the latter is preferentially expressed in sample B and not in sample A. This pattern is representative of many neural-restricted 3′ UTR extensions of genes that are expressed more broadly. These APA isoforms can be analyzed at the level of individual genes using Northern probes or RT-PCR primers directed at proximal and distal regions of the 3′ UTR; the former is “universal” since it detects both isoforms, whereas the “extension” reagents specifically recognize the long 3′ UTR. The right panels illustrate model data for Northern and qPCR measurements of APA isoforms. The middle and bottom left panels illustrate genomewide approaches, and how model data appear at specific genomic loci. RNA-seq data provides an overview of the transcriptome as reconstructed from short overlapping reads, whereas 3′-seq methods are designed to specifically capture and sequence the polyadenylated 3′ termini of mRNAs.

Figure 3. Post-transcriptional sequence elements located in 3

′ UTRs. Sequence elements located in the 3′ UTR are targets for multiple levels of post-transcriptional control. The RBP SXL binds to GU-rich DSEs to block cleavage at proximal sites, and favors generation of long APA isoforms for the e(r) gene in the Drosophila ovary. miRNAs recruit Argonaute complexes to complementary sites, mostly in 3′ UTRs, leading to decay and/or translational suppression of target transcripts. AU-rich binding proteins such as HuR and AUF1 compete for AU-rich elements to differentially regulate mRNA stability. Diverse sequence elements and structures can direct mRNA localization to subcellular regions, such as dendrites and axons. CPEB binding to targets results in cytoplasmic polyadenylation. In addition to primary sequences, secondary structures are important for binding to certain RBPs; hnRNPE1 binds a structural motif to mediate translation suppression.

Figure 4. General mechanisms of APA

(A) High levels of polyA factors, such as CstF-64, can lead to preferential usage of proximal polyA sites. (B) Lower levels of polyA factors may cause bypass of proximal sites and favor distal polyA site usage. (C) The CP machinery is associated with the carboxy-terminal domain (CTD) of RNA pol II. Slower transcriptional elongation may favor cleavage at more proximal polyA sites, whereas faster elongation rate may promote distal site usage (D). (E) RNA-binding proteins (RBPs) are thought to promote 3′ UTR lengthening by competing with the cleavage and polyadenylation (CP) machinery for cis-elements around proximal polyA sites.

Figure 5. Long 3′ UTR APA isoforms that localize to subcellular regions of neurons

bdnf expresses an extended 3′ UTR isoform that localizes to dendrites, where it undergoes localized translation. In contrast, the short 3′ UTR APA variant of bdnf is restricted to the soma. impa1 and ranbp1 express extended 3′ UTR species that are preferentially localized to axons.