tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.     

An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection.

The neural cell adhesion molecule (NCAM) gene contains an 801 nt exon that is included preferentially in neuronal cells. We have set up an in vitro splicing system that mimics the neuro-specific alternative splicing profile of NCAM exon 18. Splicing regulation is observed using model pre-mRNAs that contain competing 5' or 3' splice sites, suggesting that distinct pathways regulate NCAM 5' and 3' splice site selection. While inclusion of exon 18 is the predom-inant choice in neuronal cells, an element in the 5' common exon 17 improves exon 17/exon 19 splicing in a neuronal cell line. A similar behavior is observed in vitro as the element can stimulate the 5' splice site of exon 17 or a heterologous 5' splice site. The minimal 32 nt sequence of the exon 17 enhancer consists of purine stretches and A/C motifs. Mutations in the purine stretches compromise the binding of SR proteins and decreases splicing stimulation in vitro. Mutations in the A/C motifs do not affect SR protein binding but reduce enhancing activity. Our results suggest that the assembly of an enhancer complex containing SR proteins in a 5' common exon ensures that NCAM mRNAs lacking exon 18 are made in neuronal cells.     

Self-splicing of group II introns in vitro: lariat formation and 3' splice site selection in mutant RNAs

Deletion or substitution of the branch A residue in group II intron bl1 significantly reduces splicing activity; yet, residual exon ligation is correct, and lariats have their branch points at the normal distance from the 3' end of the intron. Mutations in the sequence facing the branch point also allow residual lariat formation; however, free 3' exons are generated with false 5' termini, all of which are within a UCACA consensus sequence located upstream or downstream of the normal 3' splice site. These results indicate that both the conserved 3' splice site APy and the spatial arrangements in stem 6 are crucial for correct 3' splice site selection.     

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.     

Spatial organization of protein-RNA interactions in the branch site-3' splice site region during pre-mRNA splicing in yeast

A series of efficiently spliced pre-mRNA substrates containing single 4-thiouridine residues were used to monitor RNA-protein interactions involving the branch site-3' splice site-3' exon region during yeast pre-mRNA splicing through cross-linking analysis. Prior to the assembly of the prespliceosome, Mud2p and the branch point bridging protein cross-link to a portion of this region in an ATP-independent fashion. Assembly of the prespliceosome leads to extensive cross-linking of the U2-associated protein Hsh155p to this region. Following the first step of splicing and in a manner independent of Prp16p, the U5 small nuclear ribonucleoprotein particle-associated protein Prp8p also associates extensively with the branch site-3' splice site-3' exon region. The subsequent cross-linking of Prp16p to the lariat intermediate is restricted to the 3' splice site and the adjacent 3' exon sequence. Using modified substrates to either mutationally or chemically block the second step, we found that the association of Prp22p with the lariat intermediate represents an authentic transient intermediate and appears to be restricted to the last eight intron nucleotides. Completion of the second step leads to the cross-linking of an unidentified approximately 80-kDa protein near the branch site sequence, suggesting a potential role for this protein in a later step in intron metabolism. Taken together, these data provide a detailed portrayal of the dynamic associations of proteins with the branch site-3' splice site region during spliceosome assembly and catalysis.     

Bimolecular exon ligation by the human spliceosome bypasses early 3' splice site AG recognition and requires NTP hydrolysis

Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3' splice site AG is supplied by a different RNA molecule than that containing the 5' splice and branch sites. By varying the time during splicing reactions when the 3' splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3' splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.     

Effect of 5'' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.     

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.     

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.     

The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites

SF2 is a 33 kd protein factor required for 5' splice site cleavage and lariat formation during pre-mRNA splicing in HeLa cell extracts. In addition to its essential role in constitutive splicing, SF2 can strongly influence 5' splice site selection. When pre-mRNAs containing multiple cis-competing 5' splice sites are spliced in vitro, high concentrations of purified SF2 promote the use of the 5' splice site closest to the 3' splice site. However, SF2 discriminates properly between authentic and cryptic splice sites. These effects of SF2 on splice site selection may reflect the cellular mechanisms that prevent exon skipping and ensure the accuracy of splicing. In addition, alterations in the concentration or activity of SF2, and of other general splicing factors, may serve to regulate alternative splicing in vivo.     

Mutational analysis of 3' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In all of the stable lines, the point mutations affected splicing efficiency in this region.     

5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.     

Intron sequences involved in lariat formation during pre-mRNA splicing

We have shown that lariat formation during in vitro splicing of several RNA precursors, from Drosophila to man, occurs at a unique and identifiable but weakly conserved site, 18 to 37 nucleotides proximal to the 3' splice site. Lariat formation within an artificial intron lacking a normal branch-point sequence occurs at a cryptic site a conserved distance (approximately 23 nucleotides) from the 3' splice site. Analysis of beta-thalassemia splicing mutations revealed that lariat formation in the first intron of the human beta-globin gene occurs at the same site in normal and mutant precursors, even though alternate 5' and 3' splice sites are utilized in the mutants. Remarkably, cleavage at the 5' splice site and lariat formation do not occur when the precursor contains a beta-thalassemia deletion removing the polypyrimidine stretch and AG dinucleotide at the 3' splice site. In contrast, a single base substitution in the AG dinucleotide blocks cleavage at the 3' splice site but not at the 5' site.     

Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5'' splice site to the internal branch acceptor site.

Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing.     

Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site.

We have introduced a single photochemical crosslinking reagent into specific sites in the central domain of U6 to identify the sites that are in close proximity to the pre-mRNA substrate. Four distinct U6 snRNAs were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively. Synthetic U6 RNA containing the 4-thioU modifications can functionally reconstitute splicing activity in cell-free yeast splicing extracts depleted of endogenous U6 snRNA. Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms crosslinks to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate. All crosslinks are dependent on the presence of ATP and the splicing substrate. The two crosslinks to the pre-mRNA from position 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splicing extracts blocked immediately prior to the first chemical step. Significantly, the crosslink from position 51 can undergo subsequent splicing when the mutant extract is complemented with functional Prp2 protein in a chase experiment, indicating that the crosslink reflects a functional interaction that is maintained during the first step. The crosslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously. This experiment is a paradigm for future studies in which different mutant extracts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur.     

U-rich tracts enhance 3' splice site recognition in plant nuclei

The process of 5' and 3' splice site definition in plant pre-mRNA splicing differs from that in mammals and yeast. In mammals, splice sites are chosen by their complementarity to U1 snRNA surrounding the /GU at the 5' splice site and by the strength of the pyrimidine tract preceding the AG/ at the 3' splice site; in plants, the 3' intron boundary is defined in a position-dependent manner relative to AU-rich elements within the intron. To determine if uridines are utilized to any extent in plant 3' splice site recognition, uridines in the region preceding the normal (−1) 3' splice site of pea rbcS3A intron 1 were replaced with adenosines. This mutant activates two cryptic 3' splice sites (+62, +95) in the downstream exon, indicating that the uridines in the region immediately preceding the normal (−1) site are essential for recognition. Placement of different length uridine tracts upstream from the cryptic +62 site indicated that a cryptic exonic 3' splice site containing 14 or 10 uridine tracts with a G at −4 can effectively outcompete the normal 3' splice site containing an eight uridine tract with a U at −4. Substitutions at the −4 position demonstrated that the identity of the nucleotide at this position greatly affects 3' splice site selection. It has been concluded that several factors affect competition between these 3' splice sites. These factors include the position of the AU transition point, the strength of the uridine tract immediately preceding the 3' terminal CAG/ and the identity of nucleotide −4.     

RNA sequences upstream of the 3' splice site repress splicing of mutantyeast ACT1 introns.

A yeast ACT1 intron in which both the first and last intron nucleotides are mutated, the /a-c/ intron, splices 10% as well as wild type. We selected for additional cis-acting mutations that improve the splicing of /a-c/ introns and recovered small deletions upstream of the 3' splice site. For example, deletion of nucleotides -9 and -10 upstream of the 3' splice site increased the splicing activity of the /a-c/ intron to 30% that of the wild-type ACT1 intron. To determine if the increased /a-c/ splicing was due to changes in intron spacing or sequence, we made mutations that mimicked the local sequence of the delta-9, -10 deletion without deleting any nucleotides. These mutants also increased /a-c/ splicing, indicating that the increased splicing activity was due to changes in intron sequence. The delta-9, -10 deletion was not allele specific to the /a-c/ intron, and improved the splicing efficiency of many mutant introns with step II splicing defects. To further define the sequences required for improved splicing of mutant introns, we randomized the region upstream of the ACT1 3' splice site. We found that almost all sequence alterations improved the splicing of the /a-c/ intron. We postulate that this sequence near the 3' end of the intron represses the splicing of mutant introns, perhaps by serving as the binding site for a negative splicing factor.