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PubMed1992

Evidence for a HeLa cell splicing activity that is necessary for activation of a regulated adenovirus 3' splice site.

Adenovirus late region 1 pre-mRNA splicing is temporally regulated during a lytic infection at the level of alternative 3' splice site usage to produce two mRNAs; the 52,55K and IIIa mRNAs which utilize the proximal and distal 3' splice sites, respectively. In vivo, the 52,55K mRNA is produced both early and late after infection, while IIIa is produced exclusively late in infection. Uninfected HeLa cell nuclear extracts, prepared with a low salt (0.4-0.5 M) or high salt (0.6 M and higher) wash, differed in their ability to splice 52,55K, IIIa and beta-globin transcripts. 52,55K and beta-globin precursors were spliced with similar efficiency in the low and high salt extract, while the IIIa mRNA was generated only in the high salt extract. Using the beta-globin pre-mRNA, no kinetic differences between the two types of extracts were observed. Nor were there any significant differences in the snRNA composition. The IIIa splicing activity did not appear to correlate with U2AF and pPTB levels. Our results suggest that a cellular trans-acting factor(s), which is required for adenovirus IIIa 3' splice site activation, is solubilized only at high salt concentrations.

Adenoviruses, Human↗
PubMed2000

The splicing determinants of a regulated exon in the axonal MAP tau reside within the exon and in its upstream intron.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile is distinct from that of the other tau regulated exons, implying the utilization of distinct regulatory factors. Previous work had established the use of cryptic splice sites within exon 6 and the influence of flanking exons on the ratio of exon 6 variants. The present work shows that, in addition to the previously identified participants, the ratio of exon 6 isoforms is affected by: (1) suppression of the cryptic sites, (2) deletions of the upstream intron, and (3) the splicing regulators PTB and U2AF, both of which act on the branch point/polypyrimidine tract region. These results strongly suggest that factors binding immediately upstream of exon 6 are involved in regulation of this exon. They also lead to the conclusion that splicing of exon 6 is primarily governed by multiple branch points.

Animals↗
PubMed1999

Differential expression of the splicing regulatory factor genes during two-step chemical transformation in a BALB/3T3-derived cell line, MT-5.

Although the alternative splicing of various genes is a common event in human tumors, the mechanisms behind it have not been characterized. We hypothesized that the expression of splicing regulatory factors would be changed during cellular transformation. Gene expression of three splicing regulatory factors, alternative splicing factor/splicing factor 2 (ASF/SF2), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 65 kDa subunit of U2 small nuclear ribonucleoprotein particles auxiliary factor (U2AF(65)), were examined by northern blotting in a two-step chemical transformation model. This in vitro model is composed of BALB/3T3 cells and a BALB/3T3-derived N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated cell line (MT-5). MT-5 cells can be transformed on exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). ASF/SF2 mRNA levels were decreased 2-fold in both MNNG-initiated cells and TPA-induced transformed cells compared with the normal parental cells, whereas hnRNP A2 mRNA expression did not significantly change between these three types of cells. U2AF(65) mRNA levels were markedly increased ( approximately 4.7-fold) associated with progression of cellular transformation. Moreover, RT-PCR analysis showed that distinct forms of ASF/SF2 mRNA were present in the MNNG-initiated cells and TPA-induced transformed cells but not in the parental cells. These findings indicate that ASF/SF2 or U2AF(65) gene expression is altered during in vitro two-step chemical transformation. The data suggest that the differential expression of splicing regulatory factors is one cause of aberrant expression of alternatively spliced mRNAs encoded by various genes in tumor cells.

3T3 Cells↗
PubMed1996

Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

Adenosine Triphosphate↗
PubMed2005

Kaposi's sarcoma-associated herpesvirus K8beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes K8alpha (K-bZIP) to induce p21 and p53 and blocks K8alpha-CDK2 interaction.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a lymphotropic DNA tumor virus that induces Kaposi's sarcoma and AIDS-related primary effusion lymphoma. KSHV open reading frame 50 and K8 genes in early viral lytic infection express, respectively, a tricistronic and a bicistronic pre-mRNA, which undergo alternative splicing to create two major spliced mRNA isoforms, alpha and beta, by inclusion (beta) or exclusion (alpha) of an intron at nucleotides 75563 to 75645. This intron contains some suboptimal features, which cause the intron 5' splice site (ss) to interact weakly with U1 snRNA and the 3' ss to bind a U2 auxiliary factor, U2AF, with low affinity. Optimization of this intron in K8 (K8 intron 2) promoted the interaction of the 5' ss with U1 and the 3' ss with U2AF, resulting in a substantial increase in intron splicing. Splicing of K8 intron 2 has also been shown to be stimulated by the splicing of a downstream intron. This was confirmed by the insertion of a human beta-globin intron into the K8beta exon 3-exon 4 splice junction, which promoted splicing of K8beta intron 2 and conversion of the K8beta mRNA to the K8alpha mRNA that encodes a K-bZIP protein. Intron 2 contains a premature termination codon, yet the K8beta mRNA is insensitive to nonsense-mediated mRNA decay, suggesting that the truncated K8beta protein may have a biological function. Indeed, although the truncated K8beta protein is missing only a C-terminal leucine zipper domain from the K-bZIP, its expression antagonizes the ability of the K-bZIP to induce p53 and p21 and blocks K-bZIP-CDK2 interaction through interfering K8alpha mRNA production.

Alternative Splicing↗
PubMed2002

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.

Adenosine Triphosphatases↗
PubMed1989

Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA branch site is an early step in spliceosome assembly and appears to commit a pre-mRNA to the splicing pathway. We have shown previously that this ATP-dependent binding requires a non-rnRNP factor, U2 snRNP auxiliary factor (U2AF), in addition to U2 snRNP. In this report we have identified U2AF, purified it to homogeneity, and characterized its biochemical properties. Purified U2AF comprises roughly equimolar quantities of two polypeptides, approximately 65 kDa and approximately 35 kDa, which appear to be associated. Measured by ultraviolet crosslinking, the 65-kDa polypeptide binds specifically to the polypyrimidine tract/3' splice site region. U2AF binds rapidly at 4 degrees C in the absence of ATP and remains associated with the pre-mRNA following U2 snRNP binding. Thus, the simple binding of U2AF initiates mammalian spliceosome assembly by facilitating the ATP-dependent binding of U2 snRNP.

Adenosine Triphosphate↗
PubMed2001

Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex.

Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a "maxi-K homology" (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by "simple" KH domains, involving residues from two putative alpha helices, a highly conserved loop, and parts of a beta sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323-332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites.

Algorithms↗
PubMed1991

The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.

Animals↗
PubMed1997

Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal.

The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF65 to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.

Animals↗
PubMed2002

New components of the spliced leader RNP required for nematode trans-splicing.

Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5' exon, the SL, is affixed to the 5'-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP). Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (M(r) 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL trans-splicing; however, neither protein is required either for conventional cis-splicing or for bimolecular (trans-) splicing of fragmented cis constructs. The M(r) 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL trans-splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a trans-splicing specific network of protein protein interactions analogous to the U1 snRNP SF1/BBP U2AF complex that comprises the cross-intron bridge in cis-splicing.

Animals↗
PubMed2003

UAP56 levels affect viability and mRNA export in Caenorhabditis elegans.

Expression of a gfp transgene in the intestines of living Caenorhabditis elegans has been measured following depletion by RNAi of a variety of known splicing factors and mRNA export proteins. Reduction of most splicing factors showed only a small effect on expression of the transgene in the animal injected with dsRNA, although most of these RNAi's resulted in embryonic lethality in their offspring. In contrast, RNAi of nxf-1, the worm homolog of mammalian NXF1/TAP, a key component of the mRNA export machinery, resulted in dramatic suppression of GFP expression in the injected animals. When we tested other proteins previously reported to be involved in marking mRNAs for export, we obtained widely divergent results. Whereas RNAi of the worm REF/Aly homologs had no obvious effect, either in the injected animals or their offspring, RNAi of UAP56, reported to be the partner of REF/Aly, resulted in strong suppression of GFP expression due to nuclear retention of its mRNA. Overexpression of UAP56 also resulted in rapid loss of GFP expression and lethality at all stages of development. We conclude that UAP56 plays a key role in mRNA export in C. elegans, but that REF/Aly may not. It also appears that some RNA processing factors are required for viability (e.g., U2AF, PUF60, SRp54, SAP49, PRP8, U1-70K), whereas others are not (e.g., U2A', CstF50).

Adenosine Triphosphatases↗
PubMed1995

A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.

Animals↗
PubMed1996

A family of novel DNA-binding nuclear proteins having polypyrimidine tract-binding motif and arginine/serine-rich motif.

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220alpha and NP220beta) have all functional domains mentioned above while two shorter forms (NP220gamma and NP220delta) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3'-splicing site of specific genes in response to differentiation-dependent signals.

Amino Acid Sequence↗
PubMed1996

Factors involved in the activation of pre-mRNA splicing from downstream splicing enhancers.

The excision of introns with weak polypyrimidine tracts at their 3' splice sites can be enhanced by sequence elements in the downstream exon or by a downstream 5' splice site. The enhancers inside the exon do not conform to a strict consensus, but they are generally rich in purines. Here, we show that members of the family of SR proteins recognize these elements. Not only does SF2/ASF activate many different polypurine enhancers, but also at least one other SR protein, most likely SC35, is active as well. The degree of splicing activation varies with the polypurine enhancers and the SR proteins. Further, we show that the similar activation by downstream 5' splice sites requires U1 snRNP, which is not the case with purine-rich enhancers. These results are consistent with a model showing that U1 snRNP binds to the 5' splice site and SR proteins to exonic sequences upstream of the 5' splice site. Both interact with U2AF at the 3' splice site. This represents a molecular explanation for the exon recognition which is important for splice site selection in mammals.

Alpharetrovirus↗
PubMed1992

Differential ASF/SF2 activity in extracts from normal WI38 and transformed WI38VA13 cells.

The normal human fibroblast cell line WI38 and a transformed derivative, WI38VA13, differentially splice fibronectin pre-mRNA in vivo. As a first step to understand the molecular basis for this regulation of splicing, we examined the ability of WI38 and WI38VA13 nuclear extracts to splice model adenovirus and globin pre-mRNAs. Adenovirus RNA splicing was detected in WI38VA13 but not in WI38 extracts. Likewise, when supplemented with a HeLa post-nuclear supernatant (S100), human beta-globin RNA splicing was detected in WI38VA13 but not in WI38 extracts. The splicing defect in WI38 extracts was associated with a reduced ability to form splicing complexes and with a corresponding decrease in the interaction of U2 small nuclear ribonucleoprotein (snRNP) with the branchsite. These defects did not correlate with a decrease in 65 kD U2AF binding since equivalent U2AF level and activity were detected in WI38 and WI38VA13 extracts. Rather, WI38 extracts displayed reduced ASF/SF2 activity and contained a low level of 30 and 40 kD SR phosphoproteins. Moreover, addition of purified ASF/SF2 dramatically increased splicing complex formation in WI38 extracts. These results raise the possibility that variations in the level and activity of ASF/SF2 and other SR proteins play a role in the regulation of fibronectin splicing.

Alternative Splicing↗