A comprehensive analysis of 3′ end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation

Abstract

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3′ untranslated region (3′ UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3′ end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3′ UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3′ end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3′ end processing in both mouse and human. With 3′ end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3′ UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3′ UTR–dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3′ end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3′ end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle.

The identification and functional annotation of RNA structures conserved in vertebrates

“…The CRSs originated from the 3 ′ end of the brain-specific mRNA KCNG2, the short 5 ′ UTR of brain-specific EOMES, lncRNA MIR4697HG (the hosted microRNA was not probed), and CRS candidate M1695693, found downstream from the annotated 3 ′ end of HOMER2 (a postsynaptic density scaffolding protein). Poly(A) signals from RNA-seq (Gruber et al 2016) supported an extended 3 ′ UTR of HOMER2 covering the CRS region (Fig. 4A).…”

“…It overlaps a DNase hypersensitive site (DHS) (ENCODE) and has the typical chromatin signatures of enhancers, namely, enrichment of H3K4me1 and reduced enrichment of H3K4me3, all indicators for a transcribed regulatory region. However, CAGE data from FANTOM5 did not support this hypothesis; instead, poly(A) site clusters (Gruber et al 2016) suggest an extended 3 ′ UTR of HOMER2. (A) Genomic tracks.…”

“…Figure 6C shows a bidirectionally transcribed locus of unknown function at the promoter of a lncRNA. Active poly(A) site data (Gruber et al 2016) revealed 2885 mRNAs and 1260 lncRNAs with an alternative poly(A) site between 50 bp and 2 kb downstream from the most distal GENCODE annotated 3 ′ end.…”

“…We considered only genes of >10 kb away from their adjacent annotated genes (∼13,000 genes). For more stringent definition of regulatory regions, taking experimentally measured transcript boundaries into account, we used the following data: DNase I hypersensitivity peak clusters from ENCODE (95 cell types) (The ENCODE Project Consortium 2009), CAGE expression of robust (>10 TPM) peaks (length ≤200 bp) from FANTOM5 Phase 2.0 (Andersson et al 2014a), ENCODE chromatin segmentation states, GENCODE gene/TSS annotation of mRNAs and lncRNAs, and polyadenylation signals (Gruber et al 2016). TSS upstream transcription and enhancers were defined by CAGE peaks, DHSs, and chromatin states.…”

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

“…A difficulty in such an approach is the recently discovered heterogeneity of polyadenylation sites that suggests the DUX4 mRNA could use alternative PAS if the major one on 4qA becomes unavailable [75,76]. Alternative polyadenylation generates 3′ UTRs of different lengths: these can recruit different factors that can impact on mRNA localization, mRNA and protein abundance, and protein intracellular location [77,78].…”