Identification of a human protein that recognizes the 3'' splice site during the second step of pre-mRNA splicing.

Accurate splicing of precursor mRNAs (pre-mRNAs) requires recognition of the 5' and 3' splice sites at the intron boundaries. Interactions between several splicing factors and the 5' splice site, which occur prior to the first step of splicing, have been well described. In contrast, recognition of the 3' splice site, which is cleaved during the second catalytic step, is poorly understood, particularly in higher eukaryotes. Here, using site-specific photo-crosslinking, we find that the conserved AG dinucleotide at the 3' splice site is contacted specifically by a 70 kDa polypeptide (p70). The p70-3' splice site crosslink has kinetics and biochemical requirements similar to those of splicing, was detected only in the mature spliceosome and occurs subsequent to the first step. Thus, p70 has all the properties expected of a factor that functionally interacts with the 3' splice site during the second step of splicing. Using antisera to various known splicing factors, we find that p70 corresponds to a previously reported 69 kDa protein of unknown function associated with the Sm core domain of spliceosomal small nuclear ribonucleoproteins.     

The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.

Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.     

U2AF-homology motif interactions are required for alternative splicing regulation by SPF45

The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site-recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-A crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.     

Switch in 3' splice site recognition between exon definition and splicing catalysis is important for sex-lethal autoregulation

Maintenance of female sexual identity in Drosophila melanogaster involves an autoregulatory loop in which the protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. We have used transient transfection of Drosophila Schneider cells to analyze the role of exon 3 splice sites in regulation. Our results indicate that exon 3 repression requires competition between the 5' splice sites of exons 2 and 3 but is independent of their relative strength. Two 3' splice site AG's precede exon 3. We report here that, while the distal site plays a critical role in defining the exon, the proximal site is preferentially used for the actual splicing reaction, arguing for a switch in 3' splice site recognition between exon definition and splicing catalysis. Remarkably, the presence of the two 3' splice sites is important for the efficient regulation by SXL, suggesting that SXL interferes with molecular events occurring between initial splice site communication across the exon and the splice site pairing that leads to intron removal.     

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.     

Splicing factor SPF30 bridges an interaction between the prespliceosome protein U2AF35 and tri-small nuclear ribonucleoprotein protein hPrp3

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.     

Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal

The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine > adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.     

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.     

Drosophila sex-lethal inhibits the stable association of the 40S ribosomal subunit with msl-2 mRNA

The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires sex-lethal (SXL) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. We delineate the msl-2 mRNA sequence elements that are important for regulation by SXL and identify functionally critical sequences adjacent to regulatory SXL binding sites. We demonstrate that SXL inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster.     

Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing.

The U5 snRNA loop 1 interacts with the 5' exon before the first step of pre-mRNA splicing and with the 5' and 3' exons following the first step. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critical for the second step in yeast spliceosomes. We systematically made deletion and insertion mutations in loop 1 then monitored splicing activity and loop-exon interactions by cross-linking. Single nucleotide deletions or insertions in loop 1 permitted both steps of splicing. Larger insertions or deletions allowed the first step but progressively inhibited the second step. Analysis of selected loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5' and 3' exons. These data indicate that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3' exon is positioned on loop 1 independently of the 5' exon.     

Sex,AGility, and the regulation of alternative splicing

Alternative splicing is an important means of regulating the expression of eukaryotic genes and enhancing protein diversity. A detailed examination of the Drosophila Sex-lethal gene has led to two significant discoveries-the role of the splicing factor SPF45 in defining the site of exon ligation, and that alternative splicing can be regulated at the second step.     

An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing

Specific mechanisms must exist to ensure fidelity in selecting the AG dinucleotide that functions as the 3' splice site during the second transesterification step of splicing. Here we show that the optimal location for this AG is within a narrow distance (19 to 23 nucleotides [nt]) downstream from the branch point sequence (BPS). Contrary to previous expectations, AGs located less than 23 nt from the BPS are always recognized, even when a second AG located more optimally downstream is used in the transesterification reaction. Indeed, the AG closest to the BPS actually dictates the precise location of the AG that engages in the reaction. This mechanism, in which the AG is identified by a general localization step followed by a precise localization step, may be used to achieve fidelity while allowing flexibility in the location of 3' splice sites.     

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.     

Extensive interactions of PRP8 protein with the 5'' and 3'' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.     

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.     

An artificial riboswitch for controlling pre-mRNA splicing

Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.     

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.