Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

Despite a growing number of splicing mutations found in hereditary diseases, utilization of aberrant splice sites and their effects on gene expression remain challenging to predict. We compiled sequences of 346 aberrant 5′splice sites (5′ss) that were activated by mutations in 166 human disease genes. Mutations within the 5′ss consensus accounted for 254 cryptic 5′ss and mutations elsewhere activated 92 de novo 5′ss. Point mutations leading to cryptic 5′ss activation were most common in the first intron nucleotide, followed by the fifth nucleotide. Substitutions at position +5 were exclusively G>A transitions, which was largely attributable to high mutability rates of C/G>T/A. However, the frequency of point mutations at position +5 was significantly higher than that observed in the Human Gene Mutation Database, suggesting that alterations of this position are particularly prone to aberrant splicing, possibly due to a requirement for sequential interactions with U1 and U6 snRNAs. Cryptic 5′ss were best predicted by computational algorithms that accommodate nucleotide dependencies and not by weight-matrix models. Discrimination of intronic 5′ss from their authentic counterparts was less effective than for exonic sites, as the former were intrinsically stronger than the latter. Computational prediction of exonic de novo 5′ss was poor, suggesting that their activation critically depends on exonic splicing enhancers or silencers. The authentic counterparts of aberrant 5′ss were significantly weaker than the average human 5′ss. The development of an online database of aberrant 5′ss will be useful for studying basic mechanisms of splice-site selection, identifying splicing mutations and optimizing splice-site prediction algorithms.     

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16⁺ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of ⁹¹QYNK⁹⁴ to ⁹¹PSFW⁹⁴ displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.     

Limited complementarity between U1 snRNA and a retroviral 5′ splice site permits its attenuation via RNA secondary structure

Multiple types of regulation are used by cells and viruses to control alternative splicing. In murine leukemia virus, accessibility of the 5′ splice site (ss) is regulated by an upstream region, which can fold into a complex RNA stem–loop structure. The underlying sequence of the structure itself is negligible, since most of it could be functionally replaced by a simple heterologous RNA stem–loop preserving the wild-type splicing pattern. Increasing the RNA duplex formation between U1 snRNA and the 5′ss by a compensatory mutation in position +6 led to enhanced splicing. Interestingly, this mutation affects splicing only in the context of the secondary structure, arguing for a dynamic interplay between structure and primary 5′ss sequence. The reduced 5′ss accessibility could also be counteracted by recruiting a splicing enhancer domain via a modified MS2 phage coat protein to a single binding site at the tip of the simple RNA stem–loop. The mechanism of 5′ss attenuation was revealed using hyperstable U1 snRNA mutants, showing that restricted U1 snRNP access is the cause of retroviral alternative splicing.     

Biased exon/intron distribution of cryptic and de novo 3' splice sites

We compiled sequences of previously published aberrant 3′ splice sites (3′ss) that were generated by mutations in human disease genes. Cryptic 3′ss, defined here as those resulting from a mutation of the 3′YAG consensus, were more frequent in exons than in introns. They clustered in ~20 nt region adjacent to authentic 3′ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3′ss that were induced by mutations outside the 3′YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 3′ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3′ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro–Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3′ss. Finally, AG-creating mutations in the PPT that produced aberrant 3′ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3′ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.     

The Emergence of Alternative 3′ and 5′ Splice Site Exons from Constitutive Exons

Alternative 3′ and 5′ splice site (ss) events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3′ss and 5′ss exons. The results revealed that alternative 3′ss and 5′ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3′ss and 5′ss exons exhibit low levels of symmetry (frame-preserving), similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.     

Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.     

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence

Many splicing factors interact with both mRNA and pre-mRNA. The identification of these interactions has been greatly improved by the development of in vivo cross-linking immunoprecipitation. However, the output carries a strong sampling bias in favor of RNPs that form on more abundant RNA species like mRNA. We have developed a novel in vitro approach for surveying binding on pre-mRNA, without cross-linking or sampling bias. Briefly, this approach entails specifically designed oligonucleotide pools that tile through a pre-mRNA sequence. The pool is then partitioned into bound and unbound fractions, which are quantified by a two-color microarray. We applied this approach to locating splicing factor binding sites in and around ∼4000 exons. We also quantified the effect of secondary structure on binding. The method is validated by the finding that U1snRNP binds at the 5′ splice site (5′ss) with a specificity that is nearly identical to the splice donor motif. In agreement with prior reports, we also show that U1snRNP appears to have some affinity for intronic G triplets that are proximal to the 5′ss. Both U1snRNP and the polypyrimidine tract binding protein (PTB) avoid exonic binding, and the PTB binding map shows increased enrichment at the polypyrimidine tract. For PTB, we confirm polypyrimidine specificity and are also able to identify structural determinants of PTB binding. We detect multiple binding motifs enriched in the PTB bound fraction of oligonucleotides. These motif combinations augment binding in vitro and are also enriched in the vicinity of exons that have been determined to be in vivo targets of PTB.     

Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer

The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5′ splice site (ss) or 3′ ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3′ ss selectivity within the 3′ ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5′ ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3′ ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.     

Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5′ splice site

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5′ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5′ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5′ss sequence, most importantly between Prp8 and nucleotides at the exon–intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.     

Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5' splice site selection

RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x_S 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x_L. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x_S 5′ ss selection, leading to decreased Bcl-x_S isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x_S 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.     

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Humans have two nearly identical copies of the survival motor neuron (SMN) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5′ splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5′ ss of exon 7. We call this structure terminal stem–loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5′ ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.     

Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1

The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5′-UGUN_xUGUN_yUGU and identify a high affinity interaction (K_d ∼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3′ or 5′ terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5′ to 3′ orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.     

Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.     

The intronic splicing code: multiple factors involved in ATM pseudoexon definition

Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5′ splice site‐like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP‐binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP‐mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans‐acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.