hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA–protein complexes at several 5′ splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5′ splice site (5′ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5′ss of TSHβ exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.     

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.     

Delineation of the mechanisms of aberrant splicing caused by two unusual intronic mutations in the RSK2 gene involved in Coffin-Lowry syndrome

Coffin–Lowry syndrome (CLS) is caused by mutations in the RSK2 gene encoding a protein kinase of the Ras signalling pathway. We have studied two point mutations which cause aberrant splicing but do not concern the invariant GT or AG nucleotides of splice sites. The first, an A→G transition at position +3 of the 5′ splice site of exon 6, results in vivo and in vitro in exon skipping and premature translation termination. The natural 5′ splice site, although intrinsically weak, is not transactivated under normal conditions. Consequently, replacement of an A/U by a G/U base pairing with U1 snRNA reduces its strength below a critical threshold. The second mutation, an A→G transition 11 nt upstream of exon 5, creates a new AG near the natural 3′ splice site. In vitro this synthetic 3′ AG is used exclusively by the splicing machinery. In vivo this splicing event is also observed, but is underestimated because the resulting RSK2 mRNA contains premature stop codons which trigger the nonsense-mediated decay process. We show that a particular mechanism is involved in the aberrant splicing of exon 5, implying involvement of the natural 3′ AG during the first catalytic step and the new 3′ AG during the second step. Thus, our results explain how these mutations cause severe forms of CLS.     

Exonic Sequences in the 5′ Untranslated Region of α-Tubulin mRNA Modulate trans Splicing in Trypanosoma brucei

Previous studies have identified a conserved AG dinucleotide at the 3′ splice site (3′SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei α-tubulin 3′SS region is required to specify accurate 3′-end formation of the upstream β-tubulin gene and trans splicing of the downstream α-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3′SS identification. Our results indicate that a minimal α-tubulin 3′SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the α-tubulin 3′SS is dependent upon the presence of exon sequences. Furthermore, β-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace α-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the α-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.     

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.     

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.     

Regulation of vif mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5′ Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5′ splice site (5′ss) D1, to the first splice acceptor, 3′ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5′ss D2, which is 50 nucleotides downstream of 3′ss A1; a GGGG silencer motif proximal to 5′ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5′ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3′ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5′ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5′ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.Human immunodeficiency virus type 1 (HIV-1) Vif is a 23-kDa basic protein (4, 9) that is incorporated into virus particles during productive infection (8-10). Replication of HIV-1 in some T-cell lines is dependent on the expression of a functional Vif protein. Replication of Vif-deleted HIV-1 is restricted in these cells, which are termed nonpermissive, because of the presence of several host deaminases, the most important of which for HIV-1 replication is APOBEC3G (A3G) (25, 26). Human A3G is a single-stranded DNA deaminase that inhibits the replication of HIV-1 as well as other types of retroviruses and retrotransposons (5, 12, 17, 25, 32). HIV-1 Vif forms a complex with A3G and other cellular proteins to promote A3G ubiquitination, resulting in proteasomal degradation of A3G (1, 11, 14, 18, 26). Vif-deleted HIV-1 produced in the presence of A3G packages increased levels of A3G compared to those found in the wild type (WT) and has reduced infectivity in nonpermissive T-cell lines. This reduced infectivity in the absence of Vif has been correlated with the dC-to-dU hypermutation of newly synthesized minus-strand viral DNA by A3G (6, 13, 31, 32). However, other studies have shown that A3G is also able to restrict virus replication without hypermutating viral DNA (7, 19).It has previously been shown that the expression of Vif in infected cells is maintained at a relatively low level compared to levels of the other HIV-1 accessory proteins. One mechanism to explain this phenomenon is that Vif is degraded more rapidly than other accessory proteins by the proteasome (3). Another mechanism is that a relatively low level of vif mRNA is produced by alternative splicing (22). Alternative splicing of HIV-1 RNA results in the production of approximately 40 different mRNA species, which include three different mRNA size classes: 1.8-kb, completely spliced RNAs; 4-kb, incompletely spliced RNAs; and 9-kb, unspliced RNAs (Fig. ​(Fig.1A).1A). The 4-kb mRNA class encodes Vif, Vpr, Tat, Vpu, and Env, and the completely spliced, 1.8-kb mRNA class encodes Tat, Rev, and Nef. Unspliced viral RNA is both packaged into virions as genomic RNA and used as mRNA for Gag and Gag-Pol proteins (2, 27). As shown in Fig. ​Fig.1A,1A, four different 5′ splice donor sites (5′ss) and eight different 3′ splice acceptor sites (3′ss), which are highly conserved among group M HIV-1 strains, are used to produce alternatively spliced HIV-1 mRNAs at different levels in infected cells (22). The efficiencies with which these 5′ss and 3′ss are used are dependent on the presence of suboptimal cis splicing elements within the 5′ss and 3′ss themselves and more-distant elements, which include exonic splicing silencers, an intronic splicing silencer, and exonic splicing enhancers (ESE) (2, 15, 27).Open in a separate windowFIG. 1.HIV-1 splicing pattern and elements regulating vif mRNA splicing. (A) The conserved 5′ss (D1 to D4) and 3′ss (A1 to A7) located within the 9-kb HIV-1 genome are shown. Completely and incompletely spliced HIV-1 mRNAs (∼4 kb and ∼1.8 kb) are shown as open boxes. Spliced mRNAs are denoted by the translated open reading frame and by the exon content. The incompletely spliced mRNAs, denoted with an I, are differentiated from completely spliced mRNAs by inclusion of the intron between 5′ss D4 and 3′ss A7. Either one or both of the noncoding exons 2 and 3 (shown as gray-shaded exons) can be differentially included within all 1.8- and 4.0-kb mRNA species, with the exception of vif mRNA (1.2I) and vpr mRNA, which can include only exon 2 (1.[2].3I). LTR, long terminal repeat. (B) Three elements regulating vif mRNA splicing are shown: positively acting enhancer ESEVif, the 5′ss D2 (underlined), and a negatively acting G4 silencer motif. The locations of noncoding exon 2 and the start site for Vif protein synthesis are also shown. (C) HIV-1 5′ss D2-down mutants used in this study are shown. Sequences were aligned and compared with that of the consensus metazoan 5′ss. The sequence of the ESEVif mutant used in this study is also aligned and compared with the WT sequence. nt, nucleotides.HIV-1 Vif is translated from a low-abundance, incompletely spliced mRNA resulting from splicing of HIV-1 RNA between the major splice donor site (5′ss D1) and 3′ss A1. We have demonstrated that vif mRNA splicing is tightly regulated by the presence of multiple regulatory elements (Fig. ​(Fig.1B).1B). These include a highly conserved suboptimal 5′ss (5′ss D2) 50 nucleotides downstream from 3′ss A1, an SRp75-dependent ESE (ESEVif), and a GGGG silencer element proximal to 5′ss D2 (2). Mutations within the relatively weak 5′ss D2 that increase its homology to a consensus 5′ss result in increased inclusion of the noncoding 50-nucleotide exon defined by 3′ss A1 and 5′ss D2 (exon 2), increased single-spliced vif mRNA levels and Vif expression, and an excessive splicing phenotype in which virion production is reduced to 10 to 25% of that of the WT (16). Conversely, mutations that decrease the homology of 5′ss D2 to a consensus 5′ss inhibit splicing at 3′ss A1 and exon 2 inclusion into both incompletely and completely spliced HIV-1 mRNAs as well as decreased levels of vif mRNA. Virus production, however, is not significantly affected. Mutation of ESEVif resulted in a similar phenotype. We have shown previously that increased or decreased exon 2 inclusion into spliced mRNA does not affect the stability or expression of viral mRNAs (15). Based on these results, we hypothesized that the conserved suboptimal 5′ss D2, which together with 3′ss A1 defines exon 2, and ESEVif are necessary to maintain optimal levels of Gag and Gag-Pol required for HIV-1 replication while producing sufficient Vif to overcome the cellular restriction factor A3G (2). To further test this hypothesis, we examined a panel of HIV-1 mutants producing reduced levels of Vif under permissive and nonpermissive conditions. We also investigated the long-term replication capabilities of these mutant viruses in both permissive and nonpermissive A3G-expressing T-cell lines. Mutant viruses demonstrated increasing sensitivity to A3G, which is inversely proportional to their levels of Vif expression. Our results suggest that the reason 5′ss D2 and ESEVif exist in the HIV-1 genome is to regulate the levels of vif mRNA and Vif protein in infected cells.     

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.     

Intrinsic differences between authentic and cryptic 5' splice sites

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5′ splice sites (5′ss), we systematically analyzed a set of 76 cryptic 5′ss derived from 46 human genes. These cryptic 5′ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5′ss. Statistical analysis of the strengths of the 5′ss using the Shapiro and Senapathy matrix revealed that authentic 5′ss have significantly higher score values than cryptic 5′ss, which in turn have higher values than the mutant ones. β-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the β-globin authentic and cryptic 5′ss, but not their surrounding context, determine the correct 5′ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5′ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5′ss-disrupting mutations at the mRNA level.     

Increased bone marrow interleukin-7 (IL-7)/IL-7R levels but reduced IL-7 responsiveness in HIV-positive patients lacking CD4+ gain on antiviral therapy

Background

The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Rα in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ ≤200/µl) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA≤50), 12 complete failures (CFs; HIV-RNA>1000), and 23 HIV-seronegative subjects.

Methods

We studied plasma IL-7 levels, IL-7Rα+CD4+/CD8+ T-cell proportions, IL-7Rα mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Rα mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells.

Results

Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Rα CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Rα mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04).

Conclusions

Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Rα expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor.     

AGO1 and AGO2 act redundantly in miR408-mediated Plantacyanin regulation

Background

In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 5′ end, respectively. Because most plant miRNAs have a 5′U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 5′A. AGO2 also participates in antiviral defense in association with viral siRNAs.

Principal Findings

This study reveals that miR408, which has a 5′A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 5′ nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 5′A-MIR408 and a mutant 5′U-MIR408 gene complement a mir408 mutant.

Conclusions/Significance

These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 5′ end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 5′ nucleotide.     

A Genetic Screen for Suppressors of a Mutated 5′ Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly

Many alleles of human disease genes have mutations within splicing consensus sequences that activate cryptic splice sites. In Caenorhabditis elegans, the unc-73(e936) allele has a G-to-U mutation at the first base of the intron downstream of exon 15, which results in an uncoordinated phenotype. This mutation triggers cryptic splicing at the −1 and +23 positions and retains some residual splicing at the mutated wild-type (wt) position. We previously demonstrated that a mutation in sup-39, a U1 snRNA gene, suppresses e936 by increasing splicing at the wt splice site. We report here the results of a suppressor screen in which we identify three proteins that function in cryptic splice site choice. Loss-of-function mutations in the nonessential splicing factor smu-2 suppress e936 uncoordination through changes in splicing. SMU-2 binds SMU-1, and smu-1(RNAi) also leads to suppression of e936. A dominant mutation in the conserved C-terminal domain of the C. elegans homolog of the human tri-snRNP 27K protein, which we have named SNRP-27, suppresses e936 uncoordination through changes in splicing. We propose that SMU-2, SMU-1, and SNRP-27 contribute to the fidelity of splice site choice after the initial identification of 5′ splice sites by U1 snRNP.PRE-mRNA splicing takes place in a large ribonucleoprotein complex called the spliceosome (Burge et al. 1999). Components of this splicing machinery assemble at conserved signal sequences within the pre-mRNA. The 5′ splice site consensus sequence M₋₃A₋₂G₋₁ | G₊₁U₊₂R₊₃A₊₄G₊₅U₊₆ and the 3′ splice site consensus sequence Y₋₃A₋₂G₋₁ | R₊₁ (M is either A or C; R is a purine, and Y is a pyrimidine) define the limits of the intron. Base-pairing interactions between the 5′ end of the U1 snRNA and the 5′ splice site consensus sequence occur early in spliceosome assembly. It is the nearly invariable GU dinucleotide at the first two positions of the 5′ end of the intron that defines the beginning of the intron. The 5′ consensus sequence is essential but insufficient for splice site selection, as 5′ splice sites with weaker consensus matches may require additional determinants for proper activation (Sanford et al. 2005).Mutations that disrupt the 5′ consensus splice signal can lead to genetic disease in humans (Nelson and Green 1990; Cohen et al. 1994). Approximately 15% of point mutations that cause genetic diseases affect pre-mRNA splicing consensus sequences (Krawczak et al. 1992). For some specific disease genes, as many as 50% of the known heritable alleles alter splicing (Teraoka et al. 1999; Ars et al. 2000; Roca et al. 2003; Pagenstecher et al. 2006). Among all the positions of the 5′ splice site consensus sequence, the highest proportion of human disease mutations occur at the +1G position (Buratti et al. 2007). The fidelity of pre-mRNA splice site choice is largely disrupted by this defect, since this mutation causes splicing at this site to be either abolished or outcompeted by the activation of nearby cryptic 5′ splice sites (Nelson and Green 1990; Cohen et al. 1994). Cryptic splice sites are used only when the wild-type splice donor is disrupted by mutation, as they tend to have very weak splice donor consensus sequences outside of a 5′-GU dinucleotide that defines the beginning of the intron (Roca et al. 2003). Suppression of mutations to the 5′ splice site consensus sequence in vivo has been achieved through the expression of U1 snRNAs containing compensatory base substitutions (Zhuang and Weiner 1986); however, suppression of mutations to the +1 position of the intron using reverse genetic approaches has not been successful (Newman et al. 1985; Nelson and Green 1990; Cohen et al. 1994).We have used a specific allele of the Caenorhabditis elegans unc-73 gene, e936, which contains a G-to-U mutation at the first nucleotide of intron 16 (Steven et al. 1998), as a model for studying cryptic splice site choice (Roller et al. 2000; Zahler et al. 2004). unc-73 encodes a RAC guanine nucleotide exchange factor that is expressed in neurons and is important for axon guidance (Steven et al. 1998). The e936 allele induces the use of three different cryptic 5′ splice sites (Figure 1A). Two of these 5′ splice sites, located at the −1 and +23 positions, define introns beginning with GU. The third 5′ splice site used is at the mutated wild-type (wt) position and is referred to as “wt” since splicing at this site still produces wild-type unc-73 mRNA and protein, even though the intron begins with UU (Roller et al. 2000). Use of either the −1 or the +23 cryptic site causes a shift in the reading frame and loss of gene function. In e936 animals, 90% of the stable messages of unc-73 are out-of-frame, yet the phenotype is not as severe as for other alleles in this gene. This indicates that the 10% of steady-state messages that are in frame have some functional role.Open in a separate windowFigure 1.—(A) Diagram of the unc-73 gene between exons 15 and 16. The positions of the −1 and +23 cryptic 5′ splice sites are indicated by arrows. The intronic e936 (+1G → U) point mutation is highlighted. (B) γ-³²P-labeled RT–PCR results across the cryptic splicing region of unc-73(e936) for different strains. Lanes 1, 2, and 3 are loaded with RT–PCR reactions from wild type (N2), unc-73(e936);sup-39(je5), and unc-73(e936) RNA, respectively. The lines carrying the suppressor alleles and e936 follow in lanes 4–10 as indicated. (C) The unc-73 genomic sequence from exon 15 (uppercase letters) and intron 15 (lowercase letters). The locations of the az23 and e936 mutational substitutions are indicated below. The position of the −9 cryptic splice donor activated in e936az23 is indicated by an arrow above.In a previous genetic screen for extragenic suppressors of e936 movement defects, Way and colleagues identified sup-39 (Run et al. 1996). It was subsequently shown that mutations in sup-39 alter cryptic splice site choice of e936 (Roller et al. 2000). sup-39 encodes a U1 snRNA gene with a compensatory mutation at the position that normally base pairs with the +1G. This allows sup-39 to base pair with an intron with a +1U (Zahler et al. 2004). This dominant suppressor increases usage of the mutated splice site and improves the fraction of in-frame messages from e936 from 10 to 33%, with a dramatic improvement in coordination. A similar mutant U1 snRNA suppressor with a different compensatory substitution, sup-6(st19), was found to suppress the intronic +1G to A transition of unc-13(e309) to allow for splicing at the mutated wild-type site, even though the intron begins with AU instead of GU (Zahler et al. 2004).We are interested in identifying additional factors that play a role in cryptic 5′ splice site choice. To do this, we took advantage of unc-73(e936), in which modest increases in the use of the wt splice site lead to dramatic increases in coordination, as a sensitive screen for changes in cryptic splice site choice. In this article we report that the proteins SMU-1 and SMU-2, which are nonessential factors previously shown to have a role in alternative splicing (Spartz et al. 2004), have a role in selection of cryptic 5′ splice sites. We also report the identification of a new dominant suppressor of cryptic splicing, snrp-27, which encodes a C. elegans homolog of the human tri-snRNP 27K protein.