Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.     

Biochemical and functional characterization of the cis-spliceosomal U1 small nuclear RNP from Trypanosoma brucei

Recent studies in the trypanosome system have revealed that in addition to trans splicing of a short spliced leader (SL) exon, there is also cis splicing of internal introns. It has been suggested that cis splicing requires base-pairing of U1 small nuclear RNA (snRNA) and the 5' splice site. We have cloned the gene for U1 snRNA from Trypanosoma brucei and characterized the U1 snRNP. Based on immunoprecipitation and direct mass-spectrometric protein analysis the U1 snRNP contains the common Sm core found also in the known trans-spliceosomal snRNPs U2, U4/U6, and U5. The 5' end of U1 snRNA in the U1 snRNP is accessible for and functional in specific recognition of the 5' splice site of the poly(A) polymerase intron.     

Identification of eight proteins that cross-link to pre-mRNA in the yeast commitment complex

In the yeast commitment complex and the mammalian E complex, there is an important base-pairing interaction between the 5' end of U1 snRNA and the conserved 5' splice site region of pre-mRNA. But no protein contacts between splicing proteins and the pre-mRNA substrate have been defined in or near this region of early splicing complexes. To address this issue, we used 4-thiouridine-substituted 5' splice site-containing RNAs as substrates and identified eight cross-linked proteins, all of which were identified previously as commitment complex components. The proteins were localized to three domains: the exon, the six nucleotides of the 5' ss region, and the downstream intron. The results indicate that the 5' splice site region and environs are dense with protein contacts in the commitment complex and suggest that some of them make important contributions to formation or stability of the U1 snRNP-pre-mRNA complex.     

Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a

Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A>G; g.75117A>G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C>T (g.75083C>T) and IVS28-159A>G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A>G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations.     

Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5' splice-site disruption

We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.     

Sequence contexts that determine the pathogenicity of base substitutions at position +3 of donor splice‐sites

Variations at position +3 of 5′ splice‐sites (5′ss) are reported to induce aberrant splicing in some cases but not in others suggesting that the overall nucleotidic environment can dictate the extent to which 5′ss are correctly selected. Functional studies of three variations identified in donor splice‐sites of USH2A and PCDH15 genes sustain this assumption. To gain insights into this question, we compared the nucleotidic context of U2‐dependent 5′ss naturally deviated (+3G,+3C, or+3T) from the+3A consensus with 5′ss for which a +3 variation (A>G, A>C, or A>T) was shown to induce aberrant splicing. Statistical differences were found between the two datasets, highlighting the role of one peculiar position in each context (+3G/+4A; +3C/?1G; and +3T/?1G). We provided experimental support to the biostatistical results through the analysis of a series of artificial mutants in reporter minigenes. Moreover, different 5′ end‐mutated U1 snRNA expression plasmids were used to investigate the importance of the position +3 and of the two identified compensatory positions ?1 and +4 in the recognition of 5′ss by the U1 snRNP. Overall, our findings establish general properties useful to molecular geneticists to identify nucleotide substitutions at position +3 that are more likely to alter splicing. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation

We have analyzed the pathway of mammalian spliceosome assembly in vitro using a mobility retardation assay. The binding of splicing complexes to both wild-type and mutant beta-globin pre-RNAs was studied. Three kinetically related, ATP-dependent complexes, alpha, beta, and gamma, were resolved with a wild-type beta-globin substrate. These complexes formed, both temporally and in order of decreasing mobility, alpha----beta----gamma. All three complexes contained U2 snRNA. The RNA intermediates of splicing, i.e., free 5' exon and intron lariat + 3' exon, were found predominantly in the gamma complex. The RNA products of splicing, i.e., ligated exons and fully excised intron lariat, were found in separate, postsplicing complexes which appeared to form via breakdown of gamma. Mutations of the 5' splice site, which caused an accumulation of splicing intermediates, also resulted in accumulation of the gamma complex. Mutations of the 3' splice site, which severely inhibited splicing, reduced the efficiency and altered the pattern of complex formation. Surprisingly, the analysis of double mutants, with sequence alterations at both the 5' and 3' splice sites, revealed that the 5' splice site genotype was important for the efficient formation of a U2 snRNA-containing alpha complex at the 3' splice site. Thus, it appears that a collaborative interaction between the separate 5' and 3' splice sites promotes spliceosome assembly.     

Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome.

The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing.     

Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG)(n)repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA.     

U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing

To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5' splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5' splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3' splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5' splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations--a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release.