A coactivator of pre-mRNA splicing

The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.     

The role of U5 snRNP in pre-mRNA splicing

The current model for the function of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome proposes that U5 carries binding sites for the 5' and 3' exons, allowing the spliceosome to 'tether' the 5' exon intermediate produced by the first catalytic step and align it with the 3' exon for the second step. Functional analysis of U5 snRNA in cis-spliceosomes has provided support for this model, and data from nematode and trypanosome splicing systems suggest that U5 or a U5-like snRNA performs a similar role in trans-splicing.     

Mechanisms of pre-mRNA splicing: classical versus non-classical pathways

Patients undergoing liver transplantation for hepatitis B-related liver disease are prone to recurrence. The mainstay of prophylaxis has been passive immunotherapy with hepatitis B immune globulin (HBIG). Antiviral therapy with lamivudine has proven effective in lowering hepatitis B virus (HBV) DNA and improving histology in patients with hepatitis B infection; its role in prophylaxis against hepatitis B recurrence following liver transplantation is under investigation. Viral breakthrough and resistance, however, are a significant problem with monotherapy with either HBIG or lamivudine. The efficacy of combination lamivudine/HBIG prophylaxis has not been reported. Fourteen patients underwent transplantation for decompensated liver disease owing to hepatitis B. Lamivudine (150 mg p.o./d) was begun before transplantation in 10 patients, including 4 who were HBV DNA-positive. In addition, 1 patient was HBV DNA-positive when transplanted. HBIG was given perioperatively and continued thereafter; treatment with lamivudine was maintained or initiated at the time of transplantation and continued indefinitely. The median follow-up was 387 days. Actuarial 1-year patient and graft survival was 93% (1 patient died of unrelated causes). At a median interval of 28 days following lamivudine treatment, all 5 HBV DNA-positive patients cleared HBV DNA from the serum; 1 went on to clear hepatitis B surface antigen (HBsAg), before transplantation, at day 148 of lamivudine treatment. By the highly sensitive polymerase chain reaction (PCR), at a median of 346 days (range, 130-525 days) following transplantation, all 13 surviving patients had no detectable serum HBV DNA. Lamivudine suppresses HBV replication in patients awaiting liver transplantation. At a median follow-up of 1.1 years, combination prophylaxis with lamivudine and HBIG prevented hepatitis B recurrence following liver transplantation.     

Cloning and characterization of PSF, a novel pre-mRNA splicing factor

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein (PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.     

The SR protein family: pleiotropic functions in pre-mRNA splicing

A family of proteins with arginine-serine-rich domains has recently come into the limelight of studies on the mechanisms of constitutive and regulated pre-mRNA splicing. Implicated in an ever increasing variety of functions, these proteins act as driving forces during spliceosome assembly and also play decisive roles in alternative splice-site selection, suggesting that they are crucial players in the regulation of splicing during cell differentiation and development.     

Evidence for a role for galectin-1 in pre-mRNA splicing

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.     

Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. We have purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). We identify the 50-kD protein as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and show that hrp48 recognizes specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48.     

Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.     

Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA

We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human beta-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3' splice-site selection.     

Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator

Alternative splicing of pre-mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre-mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1-v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type-specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions.     

A mechanism for unsplicing and exon skipping in human alpha- and beta-globin mutant pre-mRNA splicing

In both of normal human alpha- and beta-globin pre-mRNAs, two introns are spliced correctly, giving mRNA products of (exon 1)-(exon 2)-(exon 3). If 5'-splice site of the second intron is mutated, mRNA of the beta-globin is composed of (exon 1)-(exon 3), skipping the whole exon 2 sequence. However, if 5'-splice site of the second intron is mutated in the alpha-globin, mRNA is (exon 1)-(exon 2)-(intron 2)-(exon 3), unsplicing intron 2. In both of the alpha- and beta-globin pre-mRNAs, strengths of 3'-splice signals of the first and second introns were calculated by the quantification method reported previously, which explains a reason for exon skipping and unsplicing in terms of relative strengths of those signals.     

A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers

We find that the strength of splicing enhancers is determined by the relative activities of the bound serine-arginine (SR)-rich splicing factors, the number of SR proteins within the enhancer complex and the distance between the enhancer and the intron. Remarkably, the splicing activity of the bound SR proteins is directly proportional to the number of RS tetrapeptide sequences within the RS domain. Quantitative analysis of the effects of varying the distance between the enhancer and the intron revealed that the splicing efficiency is directly proportional to the calculated probability of a direct interaction between the enhancer complex and the 3' splice site. These data are consistent with a model in which splicing enhancers function by increasing the local concentration of SR proteins in the vicinity of the nearby intron through RNA looping.     

Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing

Serine/arginine (SR)-rich splicing factors contain an RNA binding domain and an arginine/serine (RS)-rich domain required for protein-protein interactions. In addition to their roles in the basic splicing reaction, SR proteins function as components of splicing enhancer complexes. Here, we investigate the role of RS domains in splicing enhancer function. Hybrid proteins containing RS domains fused to the MS2 RNA binding protein were tested in vitro with RNA substrates bearing an MS2 recognition sequence. These hybrid proteins activated splicing in nuclear extracts, but not in S100 extracts lacking SR proteins. However, intact recombinant SR proteins could complement the activity of the hybrid proteins in S100 extracts. These data demonstrate that RS domains function as splicing activators and suggest that the general and enhancer-dependent functions of SR proteins can be uncoupled.     

The branch site adenosine is recognized differently for the two steps of pre-mRNA splicing

The functional groups responsible for branch site identity in the two steps of pre-mRNA splicing as well as for spliceosome assembly were tested by incorporation of site-specific modifications at the branch site of a pre-mRNA. These results show that recognition of the adenosine occurs early in complex formation and that the branch site adenosine is recognized differently before the first step and for the second step. Further, direct UV cross-linking with these modified RNAs was used to identify a factor whose interaction was dependent upon the identity of the branch site nucleotide.     

Functional conservation of the human homolog of the yeast pre-mRNA splicing factor Prp17p

Splicing of pre-mRNAs involves two sequential transesterification reactions commonly referred to as the first and second steps. In Saccharomyces cerevisiae, four proteins, Prp16p, Prp17p, Prp18p, and Slu7p are exclusively required for the second step of splicing. The human homologs of Prp16p, Prp17p, and Prp18p have been identified, and the human proteins hPrp16 and hPrp18 have been shown to be required for the second step of splicing in vitro. Here we provide further evidence for the functional conservation of the second step factors between yeast and humans. Human hPrp17, which is 35% identical to the S. cerevisiae protein, is able to partially rescue the temperature-sensitive phenotype in a yeast strain where PRP17 has been knocked out, suggesting that the human and yeast proteins are functionally conserved. Overexpression of hPrp17 in the knockout yeast strain partially rescues the splicing defect seen in vitro and in vivo. In HeLa cells, hPrp17 is highly concentrated in the nuclear speckles, as is SC35 and many other splicing factors, thus providing further support that this protein also functions as a splicing factor in humans.     

Alternative splicing of the rabbit beta-globin pre-mRNA in vivo after transient transfection of myoblast and myotube cells

The relationship between preferences among alternative 5' splice sites and their sequences was investigated using as model mouse myoblasts and myotubes after transient transfection with the rabbit beta-globin gene. The preferences for the use of two different 5' splice sites, acting with different efficiencies to direct splicing in vivo are reported. The predominant selection of the upstream splice site has been shown in normal mouse myoblasts. In the case of differentiated myotubes the downstream splice was 1.4 times better used. The results indicate that there were differences in the preferences for the use of the two alternative splice sites between non-differentiated and terminally differentiated cells, within the same-cell line.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.