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Results for “RNA Splicing Factors”

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PubMed2000

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III.

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.

Amino Acid Sequence↗

PubMed1991

Sequences involved in the control of adenovirus L1 alternative RNA splicing.

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.

Adenoviridae↗

PubMed1995

Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast.

Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prp17-1, and prp18-1) are known to confer a specific block to the second chemical step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerevisiae. Previous studies showed that Prp16p and Prp18p are required solely for the second step in vitro. The RNA-dependent ATPase, Prp16p, functions at a stage in splicing when ATP is required, whereas Prp18p functions at an ATP-independent stage. Here we use immunodepletion to show that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require both Prp17p and ATP for slicing complementation, whereas extracts depleted of Slu7p require only the addition of Slu7p. These different ATP requirements suggest that Prp16p and Prp17p function before Prp18p and Slu7p. Although SLU7 encodes an essential gene product, we find that a null allele of prp17 is temperature-sensitive for growth and has a partial splicing defect in vitro. Finally, high-copy suppression experiments indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that these four factors may function within a multi-component complex that has both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.

Adenosine Triphosphate↗

PubMed2002

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.

Alternative Splicing↗

PubMed1991

Requirement of protein factors and ATP for the disassembly of the spliceosome after mRNA splicing reaction.

Pre-mRNA splicing reaction occurs in a large ribonucleoprotein complex called the spliceosome. After the splicing reaction, the spliceosome is disassembled to release the splicing products including spliced mRNA. Here we show that protein factors in a HeLa nuclear extract or a DEAE-cellulose fraction as well as ATP are required to release the splicing products form the spliceosome in which the splicing reaction has already completed.

Adenosine Triphosphate↗

PubMed2003

Nuclear speckles: a model for nuclear organelles.

Speckles are subnuclear structures that are enriched in pre-messenger RNA splicing factors and are located in the interchromatin regions of the nucleoplasm of mammalian cells. At the fluorescence-microscope level they appear as irregular, punctate structures, which vary in size and shape, and when examined by electron microscopy they are seen as clusters of interchromatin granules. Speckles are dynamic structures, and both their protein and RNA-protein components can cycle continuously between speckles and other nuclear locations, including active transcription sites. Studies on the composition, structure and behaviour of speckles have provided a model for understanding the functional compartmentalization of the nucleus and the organization of the gene-expression machinery.

Animals↗

PubMed2000

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Amino Acid Motifs↗

PubMed2006

Wilms' tumor 1 and signal transducers and activators of transcription 3 synergistically promote cell proliferation: a possible mechanism in sporadic Wilms' tumor.

Transcription factor Wilms' tumor 1 (WT1) was originally identified as a tumor suppressor for Wilms' tumor, but it is also overexpressed in a variety of cancer cells, suggesting a potential oncogenic function of WT1. It is important to understand molecular mechanisms underlying these dual functions of WT1 in tumorigenesis. In the current study, we report a synergistic role for signal transducers and activators of transcription 3 (STAT3) and WT1 in tumor development, including Wilms' tumor. STAT3 interacts with WT1 through its conserved domains both in vitro and in vivo. When STAT3 is activated, expression of WT1 enhances STAT3 transcriptional activity. Overexpression of WT1 and STAT3CA in NIH 3T3 increases the expression level of STAT3 target genes, including cyclin D1 and Bcl-xL, which results in an advantage of cell proliferation. Our results suggest that in the presence of activated STAT3, WT1 promotes cell proliferation instead of suppressing cell proliferation. Strikingly, STAT3 translocates to the nucleus and interacts with WT1 in a variety of primary Wilms' tumor cells, raising the hypothesis that WT1 and activated STAT3 in Wilms' tumor accelerate tumorigenesis.

Activating Transcription Factor 3↗

PubMed1998

Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p.

The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y. Tarn, C.-H. Hsu, K.-T. Huang, H.-R. Chen, H.-Y. Kao, K.-R. Lee, and S.-C. Cheng, EMBO J. 13:2421-2431, 1994). To identify proteins in the Prp19p-associated complex, we have isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using the ade2-ade3 sectoring system. A novel splicing factor, Snt309p, was identified through such a screen. Although the SNT309 gene was not essential for growth of Saccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature. Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p. Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis. Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly. These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.

Amino Acid Sequence↗

PubMed1994

Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114.

Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.

Amino Acid Sequence↗

PubMed2003

Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions.

Saccharomyces cerevisiae PRP17 (CDC40) encodes a second-step pre-mRNA splicing factor with a role in cell division. The functions of Prp17 in specific cell cycle transitions were examined using temperature-sensitive alleles in arrest/release experiments. We find that G(1)/S and G(2)/M transitions depend on Prp17. G(1)-synchronized prp17::LEU2 cells arrest at non-permissive temperatures as unbudded haploid cells with low levels of CLN1, CLB5 and RNR1 transcripts. This indicates a Prp17 execution point at or prior to Start. Reduced levels of alpha-tubulin protein, a mitotic spindle component, underlie the benomyl sensitivity of prp17 mutants and possibly their G(2)/M arrest. Splicing of TUB1 and TUB3 transcripts, which encode alpha-tubulin, was analyzed in prp17 and other second-step factor mutants. TUB1 splicing is inefficient in prp17, prp16 and prp22, and marginally affected in prp18, slu7-1 and psf1-1. TUB3 splicing is similarly affected. In vitro splicing with TUB3 pre-mRNA demonstrates a compromised second step in prp17::LEU2 extracts, implicating a direct role for Prp17 in its efficient splicing. Genomic replacement of an intronless TUB1 gene relieves the benomyl sensitivity of prp17 mutants; however, they remain temperature sensitive, implying multiple limiting factors for mitosis. The data suggest that integration of splicing with the cell cycle is important for G(1)/S and G(2)/M transitions.

Cell Cycle↗

PubMed2004

Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants.

Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site (BS) attacks the 5' splice site, and the second step, when the 5' exon attacks the 3' splice site, yielding mRNA and lariat-intron products. A genetic screen for suppressors of BS A-to-G mutants, which stall between the two steps, identified Prp8, the highly conserved spliceosomal factor. prp8 suppressors facilitate the second step for multiple intron mutants and interact functionally with first step suppressors, alleles of PRP16 and U6 snRNA. Genetic interactions among prp8, prp16, and U6 alleles suggest that these factors control a common stage in first-to-second step transition. We propose that mutant substrates are utilized by alteration of the equilibrium between first/second step conformations, resembling tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations. This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity.

Adenosine Triphosphatases↗

PubMed2006

Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets.

StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors.

Amino Acid Sequence↗

PubMed1995

SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.

Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.

Actins↗

PubMed2005

A complementation method for functional analysis of mammalian genes.

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.

Adenoviridae↗

PubMed2025

Alternative pre-mRNA Splicing and Gene Expression Patterns in Midbrain Lineage Cells Carrying Familial Parkinson's Disease Mutations.

Parkinson's disease (PD) arises from genetic and environmental factors. Human genetics has identified mutations in ~20 inherited familial genes linked to monogenic forms of PD. To investigate the effects of individual familial PD mutations, human pluripotent embryonic stem cells (hPSCs) carrying 12 distinct familial PD mutations were differentiated into midbrain lineage cells, including dopaminergic (mDA) neurons. Global gene expression and pre-mRNA splicing patterns were analyzed in midbrain cultures carrying pathogenic PD mutations in the PRKN, SNCA, LRRK2, PINK1, DNAJC6, FBXO7, SYNJ1, DJ1, VPS13C, ATP13A2 and GBA1 genes. This analysis revealed that these familial PD mutations lead to pre-mRNA splicing changes linked to RNA splicing factors and to pathways controlling cell projections, cytoskeleton, GTPase regulation and others. Importantly, we have also shown that subsets of these splicing changes overlap with changes found in PD patient postmortem brains. Mutation-specific pre-mRNA isoforms may function as both diagnostic biomarkers for familial PD-associated genotypes and promising therapeutic targets.

Journal Article↗

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↗

PubMed2002

Role of transcription in plasmid maintenance in the hpr1Delta mutant of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae hyperrecombination mutation hpr1Delta results in instability of sequences between direct repeats that is dependent on transcription of the repeat. Here it is shown that the HPR1 gene also functions in plasmid stability in the presence of destabilizing transcription elongation. In the hpr1Delta mutant, plasmid instability results from unchecked transcription elongation, which can be suppressed by a strong transcription terminator. The plasmid system has been used to examine in vivo aspects of transcription in the absence of Hpr1p. Nuclear run-on studies suggest that there is an increased RNA polymerase II density in the hpr1Delta mutant strain, but this is not accompanied by an increase in accumulation of cytoplasmic mRNA. Suppression of plasmid instability in hpr1Delta can also be achieved by high-copy expression of the RNA splicing factor SUB2, which has recently been proposed to function in mRNA export, in addition to its role in pre-mRNA splicing. High-copy-number SUB2 expression is accompanied by an increase in message accumulation from the plasmid, suggesting that the mechanism of suppression by Sub2p involves the formation of mature mRNA. Models for the role of Hpr1p in mature mRNA formation and the cause of plasmid instability in the absence of the Hpr1 protein are discussed.

Adenosine Triphosphatases↗