Context-specific regulation and function of mRNA alternative polyadenylation

Abstract

Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions (UTRs). The expression of alternative 3' UTR isoforms is highly cell type specific and is further controlled in a gene-specific manner by environmental cues. In this Review, we discuss how the dynamic, fine-grained regulation of APA is accomplished by several mechanisms, including cis-regulatory elements in RNA and DNA and factors that control transcription, pre-mRNA cleavage and post-transcriptional processes. Furthermore, signalling pathways modulate the activity of these factors and integrate APA into gene regulatory programmes. Dysregulation of APA can reprogramme the outcome of signalling pathways and thus can control cellular responses to environmental changes. In addition to the regulation of protein abundance, APA has emerged as a major regulator of mRNA localization and the spatial organization of protein synthesis. This role enables the regulation of protein function through the addition of post-translational modifications or the formation of protein-protein interactions. We further discuss recent transformative advances in single-cell RNA sequencing and CRISPR-Cas technologies, which enable the mapping and functional characterization of alternative 3' UTRs in any biological context. Finally, we discuss new APA-based RNA therapeutics, including compounds that target APA in cancer and therapeutic genome editing of degenerative diseases.

Fig. 1. Cleavage and polyadenylation of mRNA isoforms at their 3′ ends.

a | Alternative cleavage and polyadenylation (APA) generates mRNA isoforms that differ in their 3′ untranslated regions (UTRs). mRNA processing at intronic polyadenylation (IPA) sites generates mRNA isoforms (IPA isoforms) that encode proteins with alternative C-termini (light blue part of protein). mRNA processing at proximal or distal polyadenylation sites (PASs) in terminal exons generates mRNA isoforms with short 3′ UTRs (SU) or long 3′ UTRs (LU) that encode proteins with the same amino acid sequence. The grey boxes in the protein symbols represent protein domains. Introns are not drawn to scale. b | During transcription by RNA polymerase II (Pol II), the cleavage and polyadenylation (CPA) machinery binds the PAS hexamer motif AAUAAA and surrounding sequence elements in the nascent RNAs. Shown are CPA factors bound to the pre-mRNA (or nascent RNA) and to the C-terminal domain of Pol II while it is transcribing DNA. The different CPA protein complexes are colour-coded. Protein names or symbols (and gene symbols (in parentheses) when different) are given in the boxes. c | Sequence context of functional PASs. Endonucleolytic cleavage of the nascent RNA occurs ~20 nucleotides downstream of a PAS hexamer that is located in a suitable sequence context containing the UGUA and (G+U)-rich or U-rich sequence upstream and downstream, respectively. The colours of the RNA elements correspond to the colours of the complexes in part b that interact with them. CFI, cleavage factor I; CFII, cleavage factor II; CLP1, cleavage factor polyribonucleotide kinase subunit 1; CPSF1, CPA specificity factor subunit 1; CSTF, cleavage stimulation factor; FIP1, factor interacting with PAPOLA and CPSF1; nt, nucleotides; PAF1, RNA polymerase II-associated factor 1; PABPN1, poly(A)-binding protein 2; PAP, poly(A) polymerase; RBBP6, RB-binding protein 6; SCAF4, serine and arginine-related C-terminal domain-associated factor 4; TSS, transcription start site; WDR33, WD repeat domain 33.

Fig. 2. Overview of regulation of alternative polyadenylation by co-transcriptional and post-transcriptional mechanisms.

Alternative cleavage and polyadenylation (APA) is regulated both co-transcriptionally and post-transcriptionally. Cis-regulatory elements in the RNA and the DNA are necessary but not sufficient for cleavage and polyadenylation (CPA). As CPA occurs while RNA polymerase II (Pol II) transcribes a gene, Pol II elongation dynamics regulate APA. A selection of APA-regulating factors is given. Pol II termination factors show extensive crosstalk with RNA-binding proteins such as splicing factors and CPA factors that recognize RNA sequence elements and control where and when a pre-mRNA is cleaved and polyadenylated. Following processing of 3′ untranslated region isoforms, the expression level of individual APA isoforms is further regulated by post-transcriptional processes. Both the abundance and the activity of factors that control APA can be regulated co-transcriptionally and post-transcriptionally, often in a cell-type-specific and condition-specific manner, thereby allowing dynamic regulation of APA at individual genes. NEXT, nuclear exosome targeting (a trimeric protein complex); NXF1, nuclear RNA export factor 1; PAF1, RNA polymerase II-associated factor 1; PAXT, poly(A) tail exosome targeting (a trimeric protein complex); SCAF4, serine and arginine-related C-terminal domain-associated factor 4; SPT5, suppressor of Ty 5; SRSF, serine and arginine-rich splicing factor 3; THOC5, THO complex 5.

Fig. 3. Co-transcriptional regulators of alternative polyadenylation.

a | A roadblock in the DNA can affect RNA polymerase II (Pol II) translocation and use of alternative polyadenylation sites (PASs). The roadblock can be a DNA sequence element, such as a pause site, a structural element, such as a G-quadruplex, or a large protein that impedes Pol II elongation, thereby increasing the use of a proximal PAS. b | Cyclin-dependent kinase 12 (CDK12) is required for transcript elongation and suppression of premature cleavage and polyadenylation (CPA) of long genes. CDK12 promotes transcript elongation by phosphorylating Pol II. Loss of CDK12 predominantly affects DNA damage repair genes because of their extensive lengths and their lower U1 small nuclear RNA to PAS ratio. The lower processivity of Pol II results in premature CPA, thereby reducing expression of full-length mRNAs and protein output. c | Discovery of diverse alternative cleavage and polyadenylation (APA) regulators in a single genetic screen in Caenorhabditis elegans. unc-44 encodes the cytoskeleton protein ankyrin. Use of an intronic PAS in unc-44 generates intronic polyadenylation (IPA) mRNA isoforms, which encode ankyrin that is ubiquitously expressed, including in immature neurons. Giant ankyrin is expressed only in mature neurons, where it is generated from the full-length (FL) mRNA isoform through suppression of the intronic PAS. Worms that lack casein kinase 1δ (CK1δ; encoded by kin-20) have impaired movement owing to a block in neuron maturation that is caused by the lack of giant ankyrin expression. A screen for suppressor mutations identified 13 genes that, when knocked out, were able to restore the expression of giant ankyrin and the generation of mature neurons in kin-20-KO worms. The screen identified mutations that disrupt the intronic PAS sequence of the ankyrin gene, and mutations in several factors that control transcript elongation, transcription termination and CPA and in enzymes that regulate their activity. 3′-seq, 3′-end sequencing; CTD, C-terminal domain; KO, knockout; LU, long 3′ untranslated region isoform; SU, short 3′ untranslated region isoform.

Fig. 4. Gene-specific regulation of alternative 3′ UTR isoforms.

a | Differential binding of factors at alternative transcription start sites can mediate gene-specific regulation of alternative polyadenylation site (PAS) use. In fly neurons, the RNA-binding protein embryonic lethal abnormal visual system (Elav) binds to specific promoters and transcription start sites (TSS) and thus controls the expression of mRNAs with longer 3′ untranslated regions (UTRs). b | Transcription factors that bind to specific enhancers regulate APA in a gene-specific manner. Signalling-induced activation of nuclear factor-κB (NF-κB) induces its binding to a specific enhancer of the phosphatase and tensin homologue gene (PTEN) in human cell lines. NF-κB activation does not change the production or stability of the PTEN mRNA, but induces a change in 3′ UTR isoform expression. Deletion of the enhancer or silencing of NF-κB impairs this signalling-induced APA change. c | Signalling-induced phosphorylation of cleavage and polyadenylation specificity factor subunit 6 (Cpsf6) increases its activity and promotes autophagy. In flies, inactivation of the mTOR pathway allows expression of two kinases (not shown) that phosphorylate Cpsf6 in the cytoplasm, thereby promoting its translocation to the nucleus and RNA-binding activity. Cpsf6 changes the APA pattern of two master regulators of autophagy, the autophagy-related protein 1 gene (Atg1) and Atg8a. Increased expression of their long 3′ UTR (LU) isoforms supports high-level protein expression, thereby inducing autophagy upon mTOR inhibition to allow intracellular nutrient uptake. 3′-seq, 3′-end sequencing; SU, short 3′ UTR.

Fig. 5. Cell-type-specific regulation of single-UTR and multi-UTR genes is accomplished by different regulatory modes.

a | Chromatin accessibility and transcription factor (TF) binding determine cell-type-specific transcription of genes whose mRNAs contain a single 3′ untranslated region (UTR) isoform (single-UTR genes). b | Multi-UTR genes are widely transcribed (like single-UTR housekeeping genes) yet change their 3′ UTR isoforms in a cell-type-specific manner. The cell-type-specific 3′ UTR landscape is determined largely by cell-type-specific expression of regulators of alternative cleavage and polyadenylation, such as cleavage and polyadenylation specificity factor subunit 5 (CPSF5; encoded by NUDT21). c | Cooperativity between a cell-type-specific 3′ UTR landscape and activation of signalling pathways or transcription factors determines phenotypic outcomes. 3′-seq, 3′-end sequencing; CPA, cleavage and polyadenylation; iPSC, induced pluripotent stem cell; LU, long 3′ UTR; OKSM, OCT4, KLF4, SOX2 and MYC; PAS, polyadenylation site; SU, short 3′ UTR.

Fig. 6. Examples of functions of alternative 3′ UTRs.

a | 3′ untranslated region (UTR)-dependent increase in protein synthesis. Cytoplasmic polyadenylation element-binding protein 2 (CPEB2) binds to the long 3′ UTR (LU) isoform of the uncoupling protein 1 gene (Ucp1), which increases translation efficiency and protein abundance of UCP1. b | 3′ UTR-dependent mRNA localization facilitates synthesis of proteins at their final destination. Shown is a general example of local protein synthesis at the synapse of neurons. c | 3′ UTR-dependent local translation in condensates enables protein complex formation. (A+U)-rich elements represent the binding sites for the RNA-binding protein TIS11B. mRNAs that contain these elements in their 3′ UTRs localize to TIS granules, whereas lack of these elements results in localization to the endoplasmic reticulum (ER) or to the cytoplasm. Local protein synthesis in TIS granules allows the formation of protein complexes that cannot be established upon translation outside this cytoplasmic condensate. Shown is the compartment-dependent assembly of the complex between CD47 and SET, which traffics CD47 more efficiently to the plasma membrane, where it represses phagocytosis. d | 3′ UTR-dependent protein complex assembly determines protein function. A change in 3′ UTR isoform expression of the gene encoding the ubiquitin ligase BIRC3 leads to assembly of a protein complex, which changes BIRC3 function in human cells. 3′-seq, 3′-end sequencing; LU, long 3′ untranslated region; SU, short 3′ untranslated region.

Fig. 7. Manipulation of alternative 3′ UTR expression.

a | Small hairpin RNAs or small interfering RNAs (siRNAs) targeting the extended part of 3′ untranslated region (UTR) isoforms exclusively downregulate the expression of long 3′ UTR (LU) isoforms. b | Antisense oligonucleotides (ASOs) that are complementary to a specific proximal polyadenylation site (PAS) prevent binding of the cleavage and polyadenylation (CPA) machinery and exclusively downregulate expression of short 3′ UTR (SU) isoforms. c | CRISPR–iPAS. An enzymatically dead Cas13 (dCas13) is targeted by a guide RNA (dCas13 RNP) to a region slightly upstream of a PAS of interest, where it prevents CPA factors from recognizing the PAS. d | CRISPRpas. An enzymatically dead Cas9 (dCas9) is targeted by a guide RNA to a region downstream of a PAS of interest, where it acts as a roadblock for RNA polymerase II (Pol II) elongation, thereby increasing CPA at the PAS. e | Permanent removal of a specific PAS or entire 3′ UTR isoforms using a pair of Cas9 RNPs to delete the region of interest. f | PAS mutagenesis through homologous recombination can be achieved by CRISPR–Cas9-mediated DNA cleavage in the presence of a homologous repair template that contains the mutation. Cas RNP, Cas protein–guide RNA complex; KO, knockout.