Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

Abstract

Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3 ′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice.

Alternative polyadenylation of mRNA precursors

“…This was observed initially in a global analysis of all transcribed sequences in human cells 37 , and a more recent study found that ∼10% of all detected 3′ UTR-APA isoforms differed significantly in abundance between nuclear and cytoplasmic fractions 36 . Although nuclear retention was reported for long isoforms containing certain cis elements in the aUTR, such as inverted Alu repeats 38 , it is still uncertain how much of the differential localization of the long isoforms is due to differences in mRNA stability rather than differences in nuclear export.…”

“…Isoforms with a long 3′ UTR tend to be more abundant in the nucleus than in the cytoplasm 36,37 . This was observed initially in a global analysis of all transcribed sequences in human cells 37 , and a more recent study found that ∼10% of all detected 3′ UTR-APA isoforms differed significantly in abundance between nuclear and cytoplasmic fractions 36 .…”

“…Consistent with this, variations in nucleosome density and levels of histone H3 Lys36 trimethyl ation — a histone modification that is enriched at gene 3′ ends — between genes expressed at different levels were found to be much greater at proximal PASs than at distal PASs 116 . Furthermore, a recent study found that a heterochromatin formation that causes Pol II pausing also promotes the usage of proximal PASs 36 . Conversely, the more open chromatin conformation during spermato genesis, as indicated by the histone H3 Lys4 trimethylation, was found to correlate with an increased usage of proximal PASs in 3′ UTRs 129 , suggesting that open chromatin allows more efficient recruitment of the polyadenylation complex and facilitates PAS usage.…”

The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity

“…Bottom: number of transcripts in each group. Nuclear/ cytoplasmic transcript level data are from Neve et al (2016).…”

The point of no return: The poly(A)-associated elongation checkpoint

“…34 However, it is still not clear whether premature termination is linked to functional recognition of poly (A) sites. 35 To understand the spatial relationship between poly(A) sites and premature termination caused by kinase inhibition, it will be necessary to accurately map both the point of termination and the co-transcriptional usage of poly(A) sites as represented by nuclear RNA rather than total RNA. The newly-developed base-pair resolution techniques for nascent elongation transcript sequencing (NETseq) and precision nuclear run-on (PRO-Seq) in human cells will help to more accurately map termination.…”