U1 snRNP-Dependent Suppression of Polyadenylation: Physiological Role and Therapeutic Opportunities in Cancer

Abstract

Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5' splice site. U1 snRNP also plays an additional, nonsplicing global function in 3' end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3' end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.

Figure 1

Modes of U1 snRNP activity in splicing and suppression of 3′ end processing. (a) Role of U1 snRNP in splicing. The 5′ end of U1 snRNA base-pairs to the 5′ splice site cotranscriptionally, to define the functional splice donor site. The process is positively and negatively modulated by splicing factors binding to exonic and intronic splicing enhancer and silencers (ESE, ISE, ESS, and ISS, resp.). (b) Role of U1A protein in suppression of IgM cleavage and polyadenylation. U1 snRNP component U1A binds to multiple motifs (blue boxes) upstream or downstream of the intronic PAS (purple box) in IgM Cμ4 = M1 intron. These sites contain the A(U/G)GCN₁₋₃C consensus motif and are similar to U1A-binding site on U1 snRNA. From the upstream sites, U1A multimers interact directly with the C-terminal domain of PAP through a binding pocket formed by the basic-residue motifs (green triangles), blocking polyadenylation. When U1A binds downstream, it prevents CstF binding to the G/U element (light purple box) and thus interferes with cleavage. (c) Role of U1 snRNP in suppression of BPV polyadenylation. U1 binds to a 5′  ss a few nucleotides upstream of the alternative PAS. Like U1A multimers, U1-70K interacts directly with the C-terminal domain of PAP through a binding pocket formed by the basic-residue motifs, blocking polyadenylation. (d) Tethered U1-70 K suppresses polyadenylation. A modified U1-MS2 snRNA is engineered to contain an MS2-binding loop in place of the U1-70 K binding loop. A U1-binding site represses polyadenylation and expression from a luciferase reporter vector when the wt U1 snRNA is expressed. The mutant U1-MS2 snRNA recapitulates PAP inhibition only when a fusion MS2-70 K protein is also coexpressed. (e) U1 adaptors recruit U1 snRNP to suppress polyadenylation. Single-stranded, bifunctional modified ASOs are used to recruit endogenous U1 to target sites within ~1 Kb region upstream of a PAS. The targeting moiety (red) base-pairs to a unique, transcript-specific sequence, while the recruiting moiety contains a consensus 5′  ss sequence to bind U1 snRNP with high affinity. The tethered U1 snRNP suppresses polyadenylation and the mRNA is then degraded by the 3′-5′ exosome. Large blue boxes represent exons/coding regions.

Figure 2

U1 snRNP suppression of cleavage and polyadenylation safeguards transcriptome integrity. (a) In normal conditions, transcription starts bidirectionally from a RNAPII promoter. Relative enrichment of PAS and depletion of U1 sites leads to short nonproductive mRNAs on the antisense strand, thus enforcing promoter directionality (described in Almada et al. 2013 [49]). In the sense strand, the presence of U1-binding motifs at splice sites and along introns recruits U1 snRNP and suppresses IPA, allowing full-length expression. Regulated IPA sites can still be expressed depending on cellular context. (b) Reduction of U1 snRNP activity to levels still compatible with efficient splicing by partial functional knockdown using U1-binding ASOs (described in Vorlová et al. [19]) or following changes in endogenous levels [50], leads to selective APA. PASs in stronger context (or suppressed by weaker U1 sites) are activated first. These likely include regulated IPA sites and tandem APA sites, a situation that reflects what may occur in proliferative/cancer cells, with the appearance of shorter average mRNAs in part due to relative shortage of available U1 snRNP. (c) Complete disruption of U1 activity by sequestering ASOs leads to loss of splicing and release of global IPA activation. Because of transcriptional directionality, earlier IPA sites get used first, resulting in massive shortening of mRNAs (described in Kaida et al. [48]). (d) When ASOs targeted to a specific 5′ ss are used, U1 binding is disrupted in that particular location but still functions normally elsewhere. The result is the selective activation of the targeted IPA site (highlighted), with expression of a truncated variant (described in Vorlová et al. [19]). Expression of other genes is not disrupted. U1 snRNP is indicated, as well as U1-targeting ASOs (red) and transcript-specific ASOs, (blue). Large blue and light blue boxes indicate exons and UTRs, respectively, while lines depict introns. PAS are depicted as purple ovals, U1 sites as red bars. Blue arrows indicate mRNA species generated in a specific context.

Figure 3

Therapeutic potential of IPA activation: induction of secreted decoy VEGFR2. An IPA site in intron 13 of VEGFR2 can be specifically and effectively activated using ASOs targeted to the 5′ ss immediately upstream, preventing U1 from binding and thus releasing suppression [19]. This leads to the expression of a variant secreted decoy VEGFR2 encoding the sole ECD. This variant can still bind VEGF or other ligands and can still dimerize with VEGF receptors, but it cannot signal. On the contrary, it leads to a blockade of VEGF signaling in targeted and surrounding cells, with dominant negative properties.