Sensitivity of splice sites to antisense oligonucleotides in vivo

A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.     

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5' splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5' splice site to several cryptic, noncanonical 3' splice sites immediately adjacent to the normal 3' splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3' splice site sequences to pair to a given 5' splice site, albeit with tight constraints for maintaining the 3' splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.     

Temperature-dependent splicing of beta-globin pre-mRNA

A T→G mutation at nucleotide 705 of human β-globin intron 2 creates an aberrant 5′ splice site and activates a cryptic 3′ splice site upstream. In consequence, the pre-mRNA is spliced via aberrant splice sites, despite the presence of the still functional correct sites. Surprisingly, when IVS2-705 HeLa or K562 cells were cultured at temperatures below 30°C, aberrant splicing was inhibited and correct splicing was restored. Similar temperature effects were seen for another β-globin pre-mRNA, IVS2-745, and in a construct in which a β-globin intron was inserted into a coding sequence of EGFP. Temperature-induced alternative splicing was affected by the nature of the internal aberrant splice sites flanking the correct sites and by exonic sequences. The results indicate that in the context of thalassemic splicing mutations and possibly in other alternatively spliced pre-mRNAs, temperature is one of the parameters that affect splice site selection.     

Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage

Recognition of 5' splice sites in pre-mRNA splicing is achieved in part by base pairing with U1 snRNA. We have used interactive suppression in the yeast Saccharomyces cerevisiae to look for other factors involved in 5' splice-site recognition. This approach identified an extragenic suppressor that activates a cryptic 5' splice site. The suppressor is a gene for U5 snRNA (snR7) with a single base mutation in a strictly conserved 9 base sequence. This suggests that U5 snRNA can play a part in determining the position of 5' splice-site cleavage. Consistent with this, we have been able to isolate other mutations in the 9 base element in U5 snRNA that specifically activate a second cryptic 5' splice site nearby.     

Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.     

In vitro splicing of mRNA precursors: 5' cleavage site can be predicted from the interaction between the 5' splice region and the 5' terminus of U1 snRNA.

Combinations of different mutations within the 5' splice region of the rabbit beta-globin large intron were analyzed for their effect on in vitro splicing. Based upon the complementarity of the 5' splice region to the 5' terminal region of the U1 snRNA, the exact location of the 5' cleavage site of different mutants could be predicted and was experimentally confirmed. These findings add further strong support to the hypothesis (1) that the exact location of the 5' cleavage site in pre-mRNA splicing of higher eukaryotes is determined by the overall 5' splice region via the complementarity to the 5' end of the U1 snRNA, and not by the strongly conserved GU dinucleotide.     

Genetic suppression of intronic +1G mutations by compensatory U1 snRNA changes in Caenorhabditis elegans

Mutations to the canonical +1G of introns, which are commonly found in many human inherited disease alleles, invariably result in aberrant splicing. Here we report genetic findings in C. elegans that aberrant splicing due to +1G mutations can be suppressed by U1 snRNA mutations. An intronic +1G-to-U mutation, e936, in the C. elegans unc-73 gene causes aberrant splicing and loss of gene function. We previously showed that mutation of the sup-39 gene promotes splicing at the mutant splice donor in e936 mutants. We demonstrate here that sup-39 is a U1 snRNA gene; suppressor mutations in sup-39 are compensatory substitutions in the 5' end, which enhance recognition of the mutant splice donor. sup-6(st19) is an allele-specific suppressor of unc-13(e309), which contains an intronic +1G-to-A transition. The e309 mutation activates a cryptic splice site, and sup-6(st19) restores splicing to the mutant splice donor. sup-6 also encodes a U1 snRNA and the mutant contains a compensatory substitution at its 5' end. This is the first demonstration that U1 snRNAs can act to suppress the effects of mutations to the invariant +1G of introns. These findings are suggestive of a potential treatment of certain alleles of inherited human genetic diseases.     

Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6. However, no factor recognizing the highly conserved 5' splice site GU has yet been identified. We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.     

Activation of a cryptic 5' splice site by U1 snRNA

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.     

Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants

The excised introns of pre-mRNAs and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is linked by a 2'-5' phosphodiester bond (RNA branch) to a single adenosine residue near the 3' end of the intron. To determine the role of the specific sequence surrounding the RNA branch, we have mutated the branch point sequence of the human beta-globin IVS1. Pre-mRNAs lacking the authentic branch point sequence are accurately spliced in vitro; processing of the mutant pre-mRNAs generates RNA lariats due to the activation of cryptic branch points within IVS1. The cryptic branch points always occur at adenosine residues, but the sequences surrounding the branched nucleotide vary. Regardless of the type of mutation or the sequences remaining within IVS1, the cryptic branch points are 22 to 37 nucleotides upstream of the 3' splice site. These results suggest that RNA branch point selection is primarily based on a mechanism that measures the distance from the 3' splice site.     

Functional studies on the ATM intronic splicing processing element

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ~40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.     

U5 snRNA interacts with exon sequences at 5' and 3' splice sites.

U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron.     

Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.     

Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue. In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.     

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5' splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5' splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5' end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.