Influences of separation and adjacent sequences on the use of alternative 5' splice sites.

Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.     

Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3' and 5' splice sites. However, it is unknown if the 3' or the 5' splice site dominates the exon recognition process. Here, we test the 3' and 5' splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3' and 5' splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3' or the 5' splice site strength alone.     

Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.     

Intronic sequences and 3'' splice sites control Rous sarcoma virus RNA splicing.

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.     

Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing.     

A mutational analysis of U12-dependent splice site dinucleotides

Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5' A residue can splice to any 3' residue, although C is preferred. A 5' G residue can splice to 3' G or U residues with a preference for G. Little or no splicing was observed to 3' A or C residues. A 5' U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5' U to 3' U produced detectable spliced products. The dependence of 3' splice site activity on the identity of the 5' residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5' splice site and the next to last position of the 3' splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3' splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3' splice site distance of 11-12 nucleotides appears to be the same for both classes.     

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.     

Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron.

mRNAs R1 and R2 of the parvovirus minute virus of mice encode the two essential viral regulatory proteins NS1 and NS2. Both RNAs are spliced between map units 44 and 46 (nucleotides 2280 and 2399); R2 RNAs are additionally spliced upstream between map units 10 and 39 (nucleotides 514 and 1989), using a nonconsensus donor and poor 3' splice site. The relative accumulation of R1 and R2 is determined by alternative splicing: there is twice the steady-state accumulation of R2 relative to that of R1 throughout viral infection, though they are generated from the same promoter and have indistinguishable stabilities. Here we demonstrate that efficient excision of the large intron to generate R2 is dependent on at least the initial presence, in P4-generated pre-mRNAs, of sequences within the downstream small intron. This effect is orientation dependent and related to the size of the intervening exon. Prior splicing of the small intron is unnecessary. Excision of the large intron is enhanced by changing its donor site to consensus, but only in the presence of the small intron sequences. Excision of the large intron is also enhanced by improving the polypyrimidine tract within its 3' splice site; however, in contrast, this change renders excision of the large intron independent of the downstream small intron. We suggest that sequences within the small intron play a primary role in efficient excision of the upstream large intron, perhaps as the initial entry site(s) for an element(s) of the splicesome, which stabilizes the binding of required factors to the polypyrimidine tract within the 3' splice site of the large intron.     

A role for exon sequences and splice-site proximity in splice-site selection

Analysis of the in vitro splicing products of RNA precursors containing tandem duplications of the 5' or 3' splice sites of human beta-globin IVS 1 revealed that exon sequences play an important role in the relative use of the duplicated sites. These studies also show that the proximity of the 5' and 3' splice sites is an important determinant in the selection of splice-sites. Deletion, substitution, or even subtle changes of exon sequences can alter the pattern of splice-site selection, and in many cases the splice site adjacent to the altered exon is not used. The relative use of the duplicated splice sites can also be altered by diluting the splicing extract, suggesting that factors involved in splice-site selection are limiting.     

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 5' splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5' splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation.     

Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation.     

Comparative analysis detects dependencies among the 5' splice-site positions

Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5' splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5' splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, -2 and +5, and -1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5' splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5' splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5' splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5' splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5' splice site.