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PubMed2025

Precursor RNA structural patterns at SF3B1 mutation sensitive cryptic 3' splice sites.

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3' splice site selection. The SF3B1 K700E mutation (lysine to glutamic acid) is common in myelodysplastic syndrome and other blood disorders. SF3B1 K700E mutants utilize novel cryptic 3' splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 cryptic 3' splice junctions with significantly altered use in SF3B1 K700E cells, termed SF3B1-sensitive cryptic 3' splice sites, and 2800 cryptic 3' splice sites used in SF3B1 wild-type, termed SF3B1-resistant. We find that SF3B1-sensitive cryptic 3' splice sites are embedded in extended polypyrimidine tracts. Furthermore, canonical splice sites paired to SF3B1-sensitive cryptic 3' splice sites are significantly weaker than canonical 3' splice sites paired to SF3B1-resistant cryptic 3' splice sites. We test whether SF3B1-sensitive splice sites are structurally different from SF3B1-resistant 3' splice sites using chemical probing. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3' splice sites is similar. However, the magnitude of accessibility differences is less in paired SF3B1-sensitive splice sites than in paired SF3B1-mutant splice sites. Additionally, SF3B1-sensitive splice junctions are more flexible than SF3B1-resistant junctions. Our results suggest that SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3' splice site recognition in the presence of SF3B1 mutation.

RNA Splicing Factors↗
PubMed1998

Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants.

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.

Alternative Splicing↗
PubMed1999

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

Carrier Proteins↗
PubMed2004

Regulation of protein diversity by alternative pre-mRNA splicing with specific focus on chondrogenesis.

Analysis of the human genome has dramatically demonstrated that the majority of protein diversity is generated by alternative splicing of pre-mRNA. This powerful and versatile mechanism controls the synthesis of functionally different protein isoforms that may be required during specific stages of development from a single gene. Consequently, ubiquitous and/or tissue-specific RNA splicing factors that regulate this splicing mechanism provide the basis for defining phenotypic characteristics of cells during differentiation. In this review, we will introduce the basic mechanisms of pre-mRNA alternative splicing, describe how this process is regulated by specific RNA splicing factors, and relate this to various systems of cell differentiation. Chondrogenesis, a well-defined differentiation pathway necessary for skeletogenesis, will be discussed in detail, with focus on some of the alternatively-spliced proteins known to be expressed during cartilage development. We propose a heuristic view that, ultimately, it is the regulation of these RNA splicing factors that determines the differentiation status of a cell. Studying regulation at the level of pre-mRNA alternative splicing will provide invaluable insights into how many developmental mechanisms are controlled, thus enabling us to manipulate a system to select for a specific differentiation pathway.

Alternative Splicing↗
PubMed2005

Multiple roles of arginine/serine-rich splicing factors in RNA processing.

SR proteins (serine- and arginine-rich proteins) are an evolutionarily conserved family consisting of essential pre-mRNA splicing factors. Since their discovery and initial characterization, roles of SR proteins in pre-mRNA splicing and in subsequent steps of post-transcriptional gene expression have expanded significantly. The current hypotheses suggest that SR proteins are multifunctional adaptor molecules that may couple distinct steps of RNA metabolism. In the present study, we will provide an overview of the roles of SR proteins in different steps of post-transcriptional gene expression.

Arginine↗
PubMed2001

Distinct changes in intranuclear lamin A/C organization during myoblast differentiation.

Intranuclear lamin foci or speckles have been observed in various cell types. In order to explore the possibility of changes in internal lamin organization during muscle differentiation, we have examined the appearance of A-type lamin speckles that associate with RNA splicing factor speckles in C2C12 myoblasts and myotubes. Lamin speckles were observed in dividing myoblasts but disappeared early during the course of differentiation in postmitotic myocytes, and were absent in myotubes and muscle fibers. However, no changes were seen in the typical peripheral organization of lamins A/C or B1 or in RNA splicing factor speckles. Lamin speckles were also absent in quiescent myoblasts but reappeared as cells were reactivated to enter the cell cycle. These changes were not observed in other quiescent cell types. Immunoblot analysis indicated that the abundance and migration of lamins A and C was not altered in differentiated myoblasts. When myotube or quiescent myoblast nuclei were extracted with nucleases and detergent, a uniformly stained internal lamina was revealed, indicating that lamins A/C were antigenically masked in these cells, probably owing to structural reorganization of the lamina during differentiation or quiescence. Our results suggest that muscle cell differentiation is accompanied by regulated rearrangements in the organization of the A-type lamins.

Animals↗
PubMed1996

The localization of sites containing nascent RNA and splicing factors.

Sites of transcription in the HeLa nucleus are not diffusely spread throughout euchromatin but are concentrated in approximately 2000 discrete sites or "transcription factories." These sites can be immunolabeled after allowing permeabilized cells to elongate nascent RNA chains in the presence of BrUTP. Splicing factors are also concentrated in a few tens of nuclear domains known as "speckles"; they are also more diffusely spread throughout euchromatin. As there has been some controversy whether the speckles are sites of transcription, we investigated the relative distributions of transcription factories and the speckles (detected using Sm autoimmune antibodies). We used conditions that minimize the redistribution of both the nascent RNA and the antigens during permeabilization and fixation and imaged the sites with high resolution using cryosections and a "confocal" microscope. The speckles contained little-if any-nascent RNA and so were usually not transcriptionally active, but they were often surrounded by a few transcription sites. Whether they are active sites of processing of RNA or merely sites where the machinery is stored remains to be established.

Binding Sites↗
PubMed1998

Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins.

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.

Adenoviridae↗
PubMed1993

Correspondence between a mammalian spliceosome component and an essential yeast splicing factor.

None of the mammalian splicing factors that have been cloned corresponds to the yeast pre-messenger RNA splicing factors, the PRP proteins. Here, a generalizable strategy was used to isolate a complementary DNA encoding the mammalian spliceosome-associated protein (SAP) SAP 62. It is demonstrated that SAP 62 is the likely functional homolog of the yeast PRP11 protein. Both PRP11 and SAP 62 associate stably with the spliceosome, contain a single zinc finger, and display significant amino acid sequence similarity. Unlike PRP11, SAP 62 contains 22 proline-rich heptapeptide repeats at the carboxyl-terminus.

Amino Acid Sequence↗
PubMed1997

Automated electron tomography of large nuclear RNP (InRNP) particles--the naturally assembled complexes of precursor messenger RNA and splicing factors.

Splicing of nuclear pre-mRNA is an important step in the regulation of gene expression as only correctly spliced mRNAs will be exported to the cytoplasm to function in protein synthesis. Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA, are packaged in multicomponent large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200S region in sucrose gradients, contain all U snRNPs required for pre-mRNA splicing and several protein splicing factors, including U2AF and the SR proteins and can thus be viewed as naturally assembled complexes of pre-mRNA and splicing factors. We have previously reconstructed the three-dimensional image of negatively stained individual lnRNP particles by automated electron tomography. The reconstruction revealed a compact structure, 50 nm in diameter, composed of four major subunits. Here we further analyzed the reconstructed models and the apparent connectivity between the subunits using a new rendering technique. The uniformity of the lnRNP particles was substantiated by measurement of the volume engulfed by their surface. This study further supports the model proposed for the packaging of nuclear pre-mRNAs in lnRNP particles, where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multiintronic pre-mRNAs, and with the fact that the splicing of multiintronic pre-mRNAs does not occur in a sequential manner.

Animals↗
PubMed1998

The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro.

BACKGROUND: During pre-mRNA splicing, dynamic rearrangement of RNA secondary structure within the spliceosome is crucial for intron recognition and formation of the catalytic core. Splicing factors belonging to the DExD/DExH-box family of RNA-dependent ATPases are thought to have a central role in directing these rearrangements by unwinding RNA helices. Proof of this hypothesis has, however, been conspicuously lacking. RESULTS: Prp16 is a DEAH-box protein that functions in the second step of splicing in vitro. Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity. This activity is independent of sequence in either the single-stranded or duplexed regions of the RNA substrate. A mutation (prp16-1) near the ATP-binding motif of Prp16 inhibits both the RNA-dependent ATPase activity and the ATP-dependent RNA unwinding activity. CONCLUSIONS: Our findings provide strong biochemical evidence that Prp16 can disrupt a duplexed RNA structure on the spliceosome. Because the purified protein lacks sequence specificity in unwinding RNA duplexes, targeting of the unwinding activity of Prp16 in the spliceosome is likely to be determined by other interacting protein factors. The demonstration of unwinding activity will also help our understanding of how the fidelity of branchpoint recognition is controlled by Prp16.

Adenosine Triphosphatases↗
PubMed1996

Mammalian splicing factor SF1 is encoded by variant cDNAs and binds to RNA.

Mammalian splicing factor SF1 consists of a single polypeptide of 75 kDa and is required for the formation of the first ATP-dependent spliceosomal complex. Three cDNAs encoding variant forms of SF1 have been isolated and four highly related cDNAs have been found in current databases. Comparison of the cDNA sequences suggests that different SF1 mRNAs are generated by alternative splicing of a common pre-mRNA. In agreement with this idea, at least three mRNAs that are differentially expressed in different cell types have been detected by northern blot analysis. All SF1 cDNAs identified encode proteins with a common N-terminal half that contains two structural motifs implicated in RNA binding (an hnRNP K homology [KH] domain and a zinc knuckle), but the proteins differ in the length of a proline-rich region and have distinct C-termini. Three SF1 isoforms expressed in insect cells via baculovirus transfer vectors show comparable activities in the assembly of a pre-splicing complex. Consistent with the presence of a KH domain and a zinc knuckle, we show that SF1 binds directly to RNA. This interaction appears to be largely sequence-independent with a preference for guanosine- and uridine-rich sequences. The KH domain of SF1 is embedded in a 160-amino acid sequence that is shared with human Sam68, a target of Src during mitosis, as well as Caenorhabditis elegans GLD-1 and mouse Qkl, both of which play roles during cellular differentiation. The presence of this shared region in SF1 suggests functions in addition to its role in pre-spliceosome assembly.

Amino Acid Sequence↗
PubMed1993

The conserved pre-mRNA splicing factor U2AF from Drosophila: requirement for viability.

The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. The dU2AF50 protein is closely related to its mammalian counterpart and contains three carboxyl-terminal ribonucleoprotein consensus sequence RNA binding domains and an amino-terminal arginine- and serine-rich (R/S) domain. Recombinant dU2AF50 protein complements mammalian splicing extracts depleted of U2AF activity. Germline transformation of Drosophila with the dU2AF50 complementary DNA rescues a lethal mutation, establishing that the dU2AF50 gene is essential for viability. R/S domains have been found in numerous metazoan splicing factors, but their function is unknown. The mutation in Drosophila U2AF will allow in vivo analysis of a conserved R/S domain-containing general splicing factor.

Amino Acid Sequence↗
PubMed2004

Newly Initiated RNA encounters a factor involved in splicing immediately upon emerging from within RNA polymerase II.

We employed RNA-protein cross-linking to map the path of the nascent RNA as it emerges from within RNA polymerase II. A UV-cross-linkable uridine analog was incorporated at two positions within the first five nucleotides of the transcript. Only the two largest subunits of RNA polymerase II cross-linked to the transcript in complexes containing 17-24-nucleotide (nt) RNAs. Extension of the RNA to 26 or 28 nt revealed an additional strong cross-link to the splicing factor U2AF65. In U17 complexes, in which the RNA is still contained within the polymerase, U2AF65 is tightly bound. In contrast, U2AF65 is more loosely bound in C28 transcription complexes, in which about 10 nt of transcript have emerged from the RNA polymerase. Cross-linking of U2AF65 to RNA in a C28 complex was eliminated by the addition of an excess of an RNA oligonucleotide containing the consensus U2AF65 binding site, but U2AF65 was not displaced by a nonconsensus RNA. These findings indicate that U2AF65 shifts from protein-protein to protein-RNA interactions as the RNA emerges from the polymerase. During transcription of one particular template at low UTP concentration, RNA polymerase II pauses just after synthesizing a transcript segment that is a U2AF65 binding site. Dwell time of the polymerase at this pause site was significantly and specifically reduced by the addition of recombinant U2AF65 to the transcription reaction. Therefore, the association of U2AF65 with RNA polymerase II may function not only to deliver U2AF65 to the nascent transcript but also to modulate efficient transcript elongation.

Cell Nucleus↗
PubMed1999

Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF(65)).

The large subunit of the human U2 small nuclear ribonucleoprotein particle auxiliary factor (hU2AF(65)) is an essential RNA-splicing factor required for the recognition of the polypyrimidine tract immediately upstream of the 3' splice site. In the present study, we determined the solution structures of two hU2AF(65) fragments, corresponding to the first and second RNA-binding domains (RBD1 and RBD2, respectively), by nuclear magnetic resonance spectroscopy. The tertiary structure of RBD2 is similar to that of typical RNA-binding domains with the beta1-alpha1-beta2-beta3-alpha2-beta4 topology. In contrast, the hU2AF(65) RBD1 structure has unique features: (i) the alpha1 helix is elongated by one turn toward the C-terminus; (ii) the loop between alpha1 and beta2 (the alpha1/beta2 loop) is much longer and has a defined conformation; (iii) the beta2 strand is (188)AVQIN(192), which was not predicted by sequence alignments; and (iv) the beta2/beta3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four beta-strands of RBD2 whereas, in contrast, they interact with beta1, beta3 and beta4, but not with beta2 or the alpha1/beta2 loop, of RBD1. The characteristic alpha1-beta2 structure of the hU2AF(65) RBD1 may interact with other proteins, such as UAP56.

Amino Acid Sequence↗
PubMed2001

Mitochondrial Mg(2+) homeostasis is critical for group II intron splicing in vivo.

The product of the nuclear MRS2 gene, Mrs2p, is the only candidate splicing factor essential for all group II introns in mitochondria of the yeast Saccharomyces cerevisiae. It has been shown to be an integral protein of the inner mitochondrial membrane, structurally and functionally related to the bacterial CorA Mg(2+) transporter. Here we show that mutant alleles of the MRS2 gene as well as overexpression of this gene both increase intramitochondrial Mg(2+) concentrations and compensate for splicing defects of group II introns in mit(-) mutants M1301 and B-loop. Yet, covariation of Mg(2+) concentrations and splicing is similarly seen when some other genes affecting mitochondrial Mg(2+) concentrations are overexpressed in an mrs2Delta mutant, indicating that not the Mrs2 protein per se but certain Mg(2+) concentrations are essential for group II intron splicing. This critical role of Mg(2+) concentrations for splicing is further documented by our observation that pre-mRNAs, accumulated in mitochondria isolated from mutants, efficiently undergo splicing in organello when these mitochondria are incubated in the presence of 10 mM external Mg(2+) (mit(-) M1301) and an ionophore (mrs2Delta). This finding of an exceptional sensitivity of group II intron splicing toward Mg(2+) concentrations in vivo is unprecedented and raises the question of the role of Mg(2+) in other RNA-catalyzed reactions in vivo. It explains finally why protein factors modulating Mg(2+) homeostasis had been identified in genetic screens for bona fide RNA splicing factors.

Alleles↗
PubMed1996

A tyrosyl-tRNA synthetase recognizes a conserved tRNA-like structural motif in the group I intron catalytic core.

The Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns, in addition to aminoacylating tRNA(Tyr). Here, we compared the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in N. crassa mt tRNA(Tyr) by constructing three-dimensional models based on chemical modification and RNA footprinting data. Remarkably, superimposition of the CYT-18 binding sites in the model structures revealed an extended three-dimensional overlap between the tRNA and the group I intron catalytic core. Our results provide insight into how an RNA-splicing factor can evolve from a cellular RNA-binding protein. Further, the structural similarities between group I introns and tRNAs are consistent with an evolutionary relationship and suggest a general mechanism for the evolution of complex catalytic RNAs.

Base Sequence↗
PubMed2006

Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

Camptothecin↗