Processing and transcriptome expansion at the mRNA 3' end in health and disease: finding the right end

Abstract

The human transcriptome is highly dynamic, with each cell type, tissue, and organ system expressing an ensemble of transcript isoforms that give rise to considerable diversity. Apart from alternative splicing affecting the "body" of the transcripts, extensive transcriptome diversification occurs at the 3' end. Transcripts differing at the 3' end can have profound physiological effects by encoding proteins with distinct functions or regulatory properties or by affecting the mRNA fate via the inclusion or exclusion of regulatory elements (such as miRNA or protein binding sites). Importantly, the dynamic regulation at the 3' end is associated with various (patho)physiological processes, including the immune regulation but also tumorigenesis. Here, we recapitulate the mechanisms of constitutive mRNA 3' end processing and review the current understanding of the dynamically regulated diversity at the transcriptome 3' end. We illustrate the medical importance by presenting examples that are associated with perturbations of this process and indicate resulting implications for molecular diagnostics as well as potentially arising novel therapeutic strategies.

Keywords: Alternative cleavage and polyadenylation (APA); Cancer; Disease; Post-transcriptional gene regulation; RNA 3′ end maturation; Transcriptome diversity.

Fig. 1

Sequence elements and protein components involved in the formation of poly(A) tails. a The specificity and efficiency of cleavage and polyadenylation are determined by the binding of multi-protein complexes to specific elements at the 3′ end of the pre-mRNA. Most pre-mRNAs contain two core elements. The canonical polyadenylation signal AAUAAA (or less frequently AUUAAA) upstream of the cleavage site is recognized by the multimeric cleavage and polyadenylation specificity factor (CPSF) consisting of at least six subunits (CPSF 160, CPSF 100, CPSF 73, CPSF 30, hFip1, and WDR33). This RNA-protein interaction determines the site of cleavage 10–30 nt downstream, preferentially immediately 3′ of a CA dinucleotide. The second canonical sequence element is characterized by a high density of G/U or U residues which is located up to 30 nt downstream of the cleavage site. This downstream sequence element (DSE) is bound by the 64-kDa subunit of the hetero-trimeric cleavage stimulating factor (CSTF) that promotes the efficiency of 3′ end processing. Furthermore, multimers of a UGUA motif are localized at variable distances upstream of the cleavage site to recruit the heterodimeric cleavage factor CFIm [19, 176]. Finally, accessory sequences can function as upstream sequence elements (USE) [17, 32, 36, 66, 67, 75, 98, 128, 129, 132, 193] to facilitate 3′ end processing by recruiting canonical 3′ end factors directly [128, 129] or by serving as an additional anchor for the (canonical) 3′ end processing machinery [36, 66]. After assembly of the basal 3′ end processing machinery, the endonucleolytic cleavage reaction is catalyzed by CPSF-73, and the cleaved mRNA is polyadenylated by nuclear poly(A)-polymerase (PAP). The binding of PABPN1 to the poly(A) tail is unstable, and upon nuclear export, PABPN1 is replaced by the cytosolic poly(A) binding protein (PABPC), which interacts with the translation initiation factor eIF4G, stimulating translation and regulating mRNA stability. Of note, in addition to histone 3′ end processing that follows a different pathway [118, 185], other “non-canonical” mechanisms of 3′ end processing exist [189]. b Transcription initiation, elongation, and termination (circular arrow) are tightly coupled to mRNA processing steps such as capping, splicing, and 3′ end processing (inner circle). Appropriate 3′ end processing is functionally interconnected with transcription and mRNA capping and splicing and impacts on post-transcriptional mechanisms (mRNA release, export, abundance, and translation). Loss- or gain-of-function of 3′ end processing thus critically interferes with other gene expression steps (modified after [34]). c Co-transcriptional mRNA processing promotes packaging of the nascent RNA transcript (formation of an “inert” RNP particle) and thus to prevent the accumulation of co-transcriptional R-loops (lower panel), which can lead to DNA-double-strand breaks and chromosomal rearrangements. Disruption of co-transcriptional RNA-processing can thus lead to genomic instability (modified after [34])

Fig. 2

Regulated 3′ end processing in disease. a In influenza A virus-infected cells, the highly abundant NS1 protein interacts with the cellular 30 kDa subunit of CPSF and PABPN1 (not shown) [25]. This prevents binding of the CPSF complex to its RNA substrates and selectively inhibits 3′ end processing and nuclear export of host pre-mRNAs. In contrast, the 3′ terminal poly(A) sequence on viral mRNAs is produced by the viral transcriptase, which reiteratively copies a stretch of four to seven uridines in the virion RNA templates. In addition, an endonuclease intrinsic to the viral polymerase cleaves cellular capped RNAs to generate capped fragments that serve as primers for the viral mRNA synthesis (“cap-snatching mechanism” [144]). Thus, by interfering with the activity of an essential 3′ end processing factor, influenza has devised an efficient way to shut off cellular gene expression and to facilitate viral gene expression [133]. b The BRCA1-associated protein BARD1 physically interacts with CSTF-50, thereby repressing the polyadenylation machinery [90]. Both, BARD1 and CSTF-50, also interact with POL2 (not shown), and BARD1 has senses sites of DNA damage and repair. The BARD1-mediated inhibition of polyadenylation thus prevents inappropriate RNA processing during transcription at such compromised sites. Challenging cells with DNA-damaging agents results in a transient inhibition of 3′ end formation by enhanced formation of a CSTF/BARD1/BRCA1 complex. A tumor-associated germline mutation in BARD1 decreases its affinity to CSTF-50 and renders the protein inactive in polyadenylation inhibition. These findings link 3′ end RNA processing with DNA repair, and loss of wild-type BARD1 could therefore lead to defective control of gene expression as a result of inappropriate polyadenylation. c In the human prothrombin (F2) mRNA, the efficiency of 3′ end processing is regulated by engagement of a highly conserved USE [36]. After induction of p38 MAPK signaling the USE-RNP architecture changes [31]; inhibitory proteins binding to this element (red) are phosphorylated and dissociate from the USE complex while stimulatory 3′ end processing components (green) are more abundantly loaded onto the USE motif. This together with an induction of the canonical 3′ end processing machinery promotes 3′ end formation, resulting in higher level of F2 mRNA and protein. This process is believed to play an important role in the deregulation of blood coagulation during septicemia but also in processes such as tumor invasion. d The poly(A) polymerase (PAP) that catalyzes the formation of the poly(A) tail can be modified by the poly(ADP-ribose) polymerase 1 (PARP1). This regulates its activity both in vitro and in vivo. During heat shock, PARP1 binds to and modifies PAP leading to inhibition of polyadenylation in a PARP1-dependent manner. The inhibition reflects a reduced RNA binding affinity of PARylated PAP and decreased PAP association with non-heat shock protein-encoding genes [44]. Interestingly, this example also suggests that there must be gene-specific regulatory mechanisms that allow selective gene expression even in conditions, in which PAP as a central enzyme is posttranslationally modified to overcome the initial eliciting event (for further examples see [72])

Fig. 3

Mechanisms involved in the regulation of the transcriptome 3′ end diversity. Alternative 3′ end cleavage and polyadenylation (APA; two transcript isoforms shown) can be regulated (1) on the level of mRNA 3′ end processing (“direct/true APA”), through (2) alternative splicing via the in- or exclusion of PASs upon intron retention or exon skipping (“splicing coupled APA”) [183], by (3) transcriptional activities (transcription initiation, elongation, or termination; “kinetic coupling”), or (4) as a result of epigenetic regulation (i.e., through histone or DNA modifications; “epigenetic APA”). Ultimately, changing APA profiles can also be caused by (5) differential RNA turnover of individual mRNA isoforms, which have been cleaved and polyadenylated at alternative PAS (“faux/indirect APA”). Of note, another interesting mechanistic combination of both splicing regulation and concomitant transcriptional activities is executed via a U1 snRNP-mediated mechanism termed “telescripting” that protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation [11, 83] (APA = alterative cleavage and polyadenylation; CTD = C-terminal domain; POL2 = RNA polymerase II)

Fig. 4

Alternative 3′ end processing modulates the temporal and spatial diversity of gene expression. a About half of the human pre-mRNAs contain (multiple) alternative poly(A) signals (PAS), which are mostly located as tandem arrays within the 3′-UTRs [173, 194]. A smaller set of pre-mRNAs bears alternative PAS within intronic or exonic regions. In both cases, endogenous and exogenous factors can modulate pre-mRNA PAS selection by interfering with constitutive and/or auxiliary 3′ end processing factors/subunits. This results in various polyadenylated mRNA isoforms that either code for identical (tandem terminal PASs) or C-terminally modified (internal PASs) proteins. Furthermore, alternatively 3′ end processed mRNAs can display different 3′UTR properties. This can affect various aspects of mRNA and/or protein fates (export, abundance, stability, localization, transport, and translation), i.e., via the in- or exclusion of regulatory elements such as microRNA target sites or binding sites for RNA binding proteins with important roles in Mendelian disease [22]. Very commonly, cellular states associated with enhanced proliferation, dedifferentiation, or cell transformation tend to produce shorter mRNA variants (cleavage and polyadenylation occurring at the proximal PAS), while differentiation shifts cleavage and polyadenylation towards production of longer mRNA variants (processed at the distal PAS). b During B cell differentiation, alternative PAS selection effects a switch of the IgM heavy chain expression from a membrane bound form (μm) to the secreted form (μs). In this example, CSTF-64 binding to the PAS of the RNA giving rise to secreted IgM (μs) is favored either by high CSTF-64 concentrations [171] or under conditions of low hnRNP F and/or low U1A concentrations [139, 177] in plasma cells (lower lane). In contrast, the high affinity site of the membrane bound form (μm) is used in B cells (upper lane) where the CSTF-64 concentration is low or when high concentrations of U1A and/or hnRNP F inhibit CSTF-64 binding to the secretory μs-specific PAS (boxes indicate exons, simplified representation)

Fig. 5

High throughput sequencing for the (multiplexed) definition of mRNA 3′ end diversity of gene expression. a Principle for the definition of the transcriptome 3′ end (“polyA-Seq”) via (multiplexed) high-throughput sequencing based on oligo(d)t primed, barcoded cDNA library generation. b Workflow showing the bioinformatical processing of the sequencing reads involving demultiplexing, filtering, and mapping for the visualization of 3′ end seq reads. c Visualization of the transcriptome 3′ end (three selected examples) by polyA-Seq compared to RNA-Seq and definition of the mRNA 3′ end by DaPars on the basis of RNA-Seq data [191]. Shown are differential changes of the mRNA 3′ end signature comparing two conditions (“condition A” and “control,” each in biological replicates (Danckwardt lab, unpublished)). The lower panel illustrates the respective RefSeq 3′UTR and the annotation of the polyA-Seq data in comparison with a differential use of PAS in DaPars