Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.
Mol Cell Biol 1999 Oct
PMID:Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner. 1049 May 95

SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated. Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins. Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other. Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes. We show here that the S. cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential. Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome. This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.
Mol Cell Biol 1999 Oct
PMID:Characterization of a protein complex containing spliceosomal proteins SAPs 49, 130, 145, and 155. 1049 Jun 18

Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. Here we show that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, our data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA.
Hum Mol Genet 1999 Dec
PMID:Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy. 1055 82

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.
Mol Cell Biol 1999 Dec
PMID:Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65). 1056 51

To explore the dynamics of snRNP structure and function, we have studied Cus1p, identified as a suppressor of U2 snRNA mutations in budding yeast. Cus1p is homologous to human SAP145, a protein present in the 17S form of the human U2 snRNP. Here, we define the Cus1p amino acids required for function in yeast. The segment of Cus1p required for binding to Hsh49p, a homolog of human SAP49, is contained within an essential region of Cus1p. Antibodies against Cus1p coimmunoprecipitate U2 snRNA, as well as Hsh155p, a protein homologous to human SAP155. Biochemical fractionation of splicing extracts and reconstitution of heat-inactivated splicing extracts from strains carrying a temperature-sensitive allele of CUS1 indicate that Cus1p and Hsh155p reside in a functional, high-salt-stable complex that is salt-dissociable from U2 snRNA. We propose that Cus1p, Hsh49p, and Hsh155p exist in a stable protein complex which can exchange with a core U2 snRNP and which is necessary for U2 snRNP function in prespliceosome assembly. The Cus1p complex shares functional as well as structural similarities with human SF3b.
Mol Cell Biol 2000 Mar
PMID:Functional Cus1p is found with Hsh155p in a multiprotein splicing factor associated with U2 snRNA. 1068 64

Childhood onset spinal muscular atrophy (SMA) is a common autosomal recessive disorder primarily characterized by the loss of lower alpha motor neurons. The underlying chromosomal defects causing SMA have been found in the survival motor neuron (SMN) gene. SMN has been shown previously to play a role in both snRNP biogenesis and mRNA processing, although direct evidence for the relationship between SMN and disease pathology has not been elucidated. SMN orthologues have been isolated in many species including Caenorhabditis elegans and Danio rerio. To study the function of SMN, we have identified and characterized the Schizosaccharomyces pombe orthologue of human SMN, smn1 (+). We have demonstrated that smn1 (+) is essential for viability in S.pombe and yeast expressing missense mutations in Smn1p, which mimic mutations in patients with Type I SMA, show significant mislocalization of the protein and a decrease in cell viability. Wild-type Smn1p is localized predominantly in the nucleus whereas yeast expressing Smn1p with missense mutations or deletions of specific domains of the protein accumulate cytoplasmic aggregates. Overexpression of Smn1p results in an increase in the growth rate of cells. Furthermore, mutations within two highly conserved protein interaction domains have a dominant-negative effect on growth, indicating that each domain is of functional significance in S.pombe. These dominant phenotypes can be suppressed by overexpression of murine Smn in the same cell. Given the structural and functional similarities between the protein in fission yeast and higher eukaryotes, S.pombe will be an ideal organism to study the role of SMN in RNA processing.
Hum Mol Genet 2000 Mar 22
PMID:Characterization of the Schizosaccharomyces pombe orthologue of the human survival motor neuron (SMN) protein. 1074 74

An essential step of pre-mRNA spliceosome assembly is the interaction between the snRNPs U4/U6 and U5, to form the [U4/U6.U5] tri-snRNP. While the tri-snRNP protein Prp6p appears to play an important role for tri-snRNP formation in yeast, little is known about the interactions that connect the two snRNP particles in human tri-snRNPs. Here, we describe the molecular characterisation of a 102kD protein form HeLa tri-snRNPs. The 102kD protein exhibits a significant degree of overall homology with the yeast Prp6p, including the conservation of multiple tetratrico peptide repeats (TPR), making this the likely functional homologue of Prp6p. However, while the yeast Prp6p is considered to be a U4/U6-specific protein, the human 102kD protein was found to be tightly associated with purified 20 S U5 snRNPs. This association appears to be primarily due to protein-protein interactions. Interestingly, antibodies directed against the C-terminal TPR elements of the 102kD protein specifically and exclusively immunoprecipitate free U5 snRNPs, but not [U4/U6.U5] tri-snRNPs, from HeLa nuclear extract, suggesting that the C-terminal region of the 102kD protein is covered by U4/U6 or tri-snRNP-specific proteins. Since proteins containing TPR elements are typically involved in multiple protein-protein interactions, we suggest that the 102kD protein interacts within the tri-snRNP with both the U5 and U4/U6 snRNPs, thus bridging the two particles. Consistent with this idea, we show that in vitro translated U5-102kD protein binds to purified 13S U4/U6 snRNPs, which contain, in addition to the Sm proteins, all known U4/U6-specific proteins.
J Mol Biol 2000 May 12
PMID:The human homologue of the yeast splicing factor prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions. 1078 20

Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.
Mol Cell Biol 2000 Jun
PMID:Aberrant splicing of tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism linked to chromosome 17. 1080 46

In the current model for spliceosome assembly, U1 snRNP binds to the 5' splice site in the E complex followed by ATP-dependent binding of U2 snRNP to the branchpoint sequence (BPS) in the A complex. Here we report the characterization of highly purified, functional E complex. We provide evidence that this complex contains functional U2 snRNP and that this snRNP is required for E complex assembly. The BPS is not required for U2 snRNP binding in the E complex. These data suggest a model for spliceosome assembly in which U1 and U2 snRNPs first associate with the spliceosome in the E complex and then an ATP-dependent step results in highly stable U2 snRNP binding to the BPS in the A complex.
Mol Cell 2000 May
PMID:Functional association of U2 snRNP with the ATP-independent spliceosomal complex E. 1088 14

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site. We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.
Mol Cell Biol 2000 Sep
PMID:The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site. 1093 5


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