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Results for “Spliceosomes”

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PubMed1993

Convergent transcripts of the yeast PRP38-SMD1 locus encode two essential splicing factors, including the D1 core polypeptide of small nuclear ribonucleoprotein particles.

The PRP38 gene of Saccharomyces cerevisiae is necessary for the excision of intron sequences from pre-mRNA and required for the maintenance of maximal levels of U6 small nuclear RNA (snRNA). This report describes the identification of a gene of related function, SMD1, located immediately 3' to PRP38. The PRP38 and SMD1 transcription units are configured in an unusual "tail-to-tail" arrangement with their respective open reading frames terminating on opposite strands of a common 6-bp region. The predicted SMD1 polypeptide, Smd1p, is 40% identical to the D1 protein of human small nuclear ribonucleoprotein particles. Experimentally induced depletion of Smd1p blocks the first step of splicing and results in growth arrest. In addition, the levels of the trimethylguanosine-capped spliceosomal snRNAs, U1, U2, U4, and U5, but not the Prp38p-sensitive U6 snRNA, decrease in response to Smd1p depletion. The cap structures of snRNAs persisting in the absence of SMD1 expression appear to be peculiar, as they are poorly recognized by an anti-trimethylguanosine antibody. These data establish Smd1p as a required component of the cellular splicing apparatus and a factor in snRNA maturation and stability.

Amino Acid Sequence↗

PubMed1997

Transient aggregates in protein folding are easily mistaken for folding intermediates.

It has been questioned recently whether populated intermediates are important for the protein folding process or are artefacts trapped in nonproductive pathways. We report here that the rapidly formed intermediate of the spliceosomal protein U1A is an off-pathway artefact caused by transient aggregation of denatured protein under native conditions. Transient aggregates are easily mistaken for structured monomers and could be a general problem in time-resolved folding studies.

Artifacts↗

PubMed1999

Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing.

The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G(2)/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.

3T3 Cells↗

PubMed2000

Evidence for a linear search in bimolecular 3' splice site AG selection.

In most eukaryotic introns the 3' splice site is defined by a surprisingly short AG consensus found a variable distance downstream of the branch site. Exactly how the spliceosome determines which AG to use, however, is not well understood. Previously we showed that when the branch site and 3' splice site AG are supplied by separate RNA molecules, there is a strong preference for use of the 5'-most AG in the 3' splice site-containing RNA. Here we show that this apparent 5'-->3' directionality holds even when this RNA contains four tandem repeats of a 6-nt sequence containing AG. Exactly the same pattern of 3' splice site choice was observed when the same tandem repeats were incorporated into a full-length splicing substrate. When the 3' splice site AG is supplied by a separate RNA, that RNA must be linear with an unobstructed 5' end. Similarly, the branch-containing RNA must be truncated immediately 3' to the polypyrimidine tract. A model is presented that incorporates these observations and reconciles previously proposed mechanisms for 3' splice site selection.

Adenine Nucleotides↗

PubMed1995

In vitro splicing deficiency induced by a C to T mutation at position -3 in the intron 10 acceptor site of the phenylalanine hydroxylase gene in a patient with phenylketonuria.

A previous study has identified a C-->U mutation at position -3 in the 3' splice site of intron 10 of the phenylalanine hydroxylase pre-mRNA in a patient with phenylketonuria. In vivo, this mutation induces the skipping of the downstream exon. This result is puzzling because both CAG and UAG have been reported to function equally as 3' splice sites. In this report, we show that the C-->U mutation affects predominantly the first step of the splicing reaction and that it blocks spliceosome assembly at an early stage. The 3' region of the phenylalanine hydroxylase intron 10 has two unusual characteristic features: multiple potential branch sites and a series of four guanosine residues, which interrupt the polypyrimidine tract at positions -8 to -11 from the 3' splice site. We show that the mutation precludes the use of the proximal branch site, while having no effect on the remote one. We also show that in the UAG transcript, the four guanosine residues inhibit the splicing of intron 10. The substitution of these purine residues by one cytosine residue, regardless of the position, increases the splicing efficiency of the mutant UAG precursor while having no effect on the wild-type CAG precursor. Substituting the four purine residues by four pyrimidines relieves the inhibition and rescues the use of the proximal branch site. These results demonstrate that according to the context, the C and U nucleotides preceding the AG are not equivalent for the splicing reaction.

Base Sequence↗

PubMed2001

Binding sites for Rev and ASF/SF2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus RNA.

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.

Alternative Splicing↗

PubMed2002

Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1.

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca(2+) chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The protein phosphatase-1 (PP1) regulator, nuclear inhibitor of PP1 (NIPP1), interacts with the polycomb group protein, embryonic ectoderm development (EED), and functions as a transcriptional repressor.

The nuclear protein NIPP1 (nuclear inhibitor of protein Ser/Thr phosphatase-1) interacts with the splicing factors SAP155 and CDC5L and is involved in a late step of spliceosome assembly. In addition, NIPP1 is an interactor of protein phosphatase-1 and a COOH-terminal NIPP1 fragment displays an RNase E like endoribonuclease activity. A yeast two-hybrid screening resulted in the identification of the Polycomb group protein EED (embryonic ectoderm development), an established transcriptional repressor, as a novel NIPP1 interactor. NIPP1 only interacted with full-length EED, whereas two EED interaction domains were mapped to the central and COOH-terminal thirds of NIPP1. The NIPP1-EED interaction was potentiated by the binding of (d)G-rich nucleic acids to the central domain of NIPP1. Nucleic acids also decreased the potency of NIPP1 as an inhibitor of PP1, but they did not prevent the formation of a ternary NIPP1.EED.PP1 complex. EED had no effect on the function of NIPP1 as a splicing factor or as an endoribonuclease. However, similar to EED, NIPP1 acted as a transcriptional repressor of targeted genes and this NIPP1 effect was mediated by the EED interaction domain. Also, the histone deacetylase 2 was present in a complex with NIPP1. Our data are in accordance with a role for NIPP1 as a DNA-targeting protein for EED and associated chromatin-modifying enzymes.

Animals↗

PubMed2003

A simple whole cell lysate system for in vitro splicing reveals a stepwise assembly of the exon-exon junction complex.

Pre-mRNA splicing removes introns and leaves in its wake a multiprotein complex near the exon-exon junctions of mRNAs. This complex, termed the exon-exon junction complex (EJC), contains at least seven proteins and provides a link between pre-mRNA splicing and downstream events, including transport, localization, and nonsense-mediated mRNA decay. Using a simple whole cell lysate system we developed for in vitro splicing, we prepared lysates from cells transfected with tagged EJC proteins and studied the association of these proteins with pre-mRNA, splicing intermediates, and mRNA, as well as formation of the EJC during splicing. Three of the EJC components, Aly/REF, RNPS1, and SRm160, are found on pre-mRNA by the time the spliceosome is formed, whereas Upf3b associates with splicing intermediates during or immediately after the first catalytic step of the splicing reaction (cleavage of exon 1 and intron-lariat formation). In contrast, Y14 and magoh, which remain stably associated with mRNA after export to the cytoplasm, join the EJC during or after completion of exon-exon ligation. These findings indicate that EJC formation is an ordered pathway that involves stepwise association of components and is coupled to specific intermediates of the splicing reaction.

Blotting, Western↗

PubMed2003

Structuring of the 3' splice site by U2AF65.

Recognition of the 3' splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3' splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3' splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA. These studies, combined with an analysis of extant high resolution x-ray structures of protein.RNA complexes, suggest a model whereby U2AF65 bends the pre-mRNA to juxtapose reactive functionalities of the pre-mRNA substrate and organize these structures for subsequent spliceosome assembly.

Binding Sites↗

PubMed2006

An exonic splicing silencer is involved in the regulated splicing of glucose 6-phosphate dehydrogenase mRNA.

The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.

Animals↗

PubMed2006

Gemin8 is required for the architecture and function of the survival motor neuron complex.

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) in higher eukaryotes requires the functions of several cellular proteins and includes nuclear as well as cytoplasmic phases. In the cytoplasm, a macromolecular complex containing the survival motor neuron (SMN) protein, Gemin2-8 and Unrip mediates the ATP-dependent assembly of Sm proteins and snRNAs into snRNPs. To carry out snRNP assembly, the SMN complex binds directly to both Sm proteins and snRNAs; however, the contribution of the individual components of the SMN complex to its composition, interactions, and function is poorly characterized. Here, we have investigated the functional role of Gemin8 using novel monoclonal antibodies against components of the SMN complex and RNA interference experiments. We show that Gemin6, Gemin7, and Unrip form a stable cytoplasmic complex whose association with SMN requires Gemin8. Gemin8 binds directly to SMN and mediates its interaction with the Gemin6/Gemin7 heterodimer. Importantly, loss of Gemin6, Gemin7, and Unrip interaction with SMN as a result of Gemin8 knockdown affects snRNP assembly by impairing the SMN complex association with Sm proteins but not with snRNAs. These results reveal the essential role of Gemin8 for the proper structural organization of the SMN complex and the involvement of the heteromeric subunit containing Gemin6, Gemin7, Gemin8, and Unrip in the recruitment of Sm proteins to the snRNP assembly pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

L4-33K, an adenovirus-encoded alternative RNA splicing factor.

Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3' splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3' splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus late-infected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.

Adenoviridae↗

PubMed2006

Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets.

Prp19p is an integral component of the heteromeric protein complex (the NineTeen complex) in the nucleus, and it is essential for the structural integrity of NineTeen complex and its subsequent activation of the spliceosome. We identified Prp19p, which has never been reported in relation to any function outside of the nucleus, as a member of proteins associated with lipid droplets. Down-regulation of Prp19p expression with RNA interference in 3T3-L1 cells repressed lipid droplet formation with the reduction in the level of expression of perilipin and S3-12. The levels of expression of SCD1 (stearoyl-CoA desaturase-1), DGAT-1 (acyl-CoA diacylglycerol acyltransferase-1), and glycerol-3-phosphate acyltransferase were also reduced in Prp19p down-regulated cells, and a significant decrease in triglycerides was observed. Unlike perilipin, which is one of the most extensively studied lipid droplet-associated proteins, Prp19p is not essential for cAMP- and hormone-sensitive lipase-dependent lipolysis pathways, even though Prp19p is a component of the lipid droplet phospholipid monolayer, and down-regulation of Prp19p represses fat accretion significantly. These results suggest that Prp19p or Prp19-interacting proteins during lipid droplet biogenesis in adipocytes may be considered as another class of potential targets for attacking obesity and obesity-related problems.

3T3-L1 Cells↗

PubMed1994

Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei.

In the interphase nucleus of mammalian cells the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear domains including interchromatin granules and coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and reassembly of daughter nuclei. At the onset of mitosis snRNPs are predominantly diffuse in the cytoplasm, although a subset remain associated with remnants of coiled bodies and clusters of mitotic interchromatin granules, respectively. The number and size of mitotic coiled bodies remain approximately unchanged from metaphase to early telophase while snRNP-containing clusters of mitotic interchromatin granules increase in size and number as cells progress from anaphase to telophase. During telophase snRNPs are transported into daughter nuclei while the clusters of mitotic interchromatin granules remain in the cytoplasm. The timing of nuclear import of splicing snRNPs closely correlates with the onset of transcriptional activity in daughter nuclei. When transcription restarts in telophase cells snRNPs have a diffuse nucleoplasmic distribution. As cells progress to G1 snRNP-containing clusters of interchromatin granules reappear in the nucleus. Coiled bodies appear later in G1, although the coiled body antigen, p80 coilin, enters early into telophase nuclei. After inhibition of transcription we still observe nuclear import of snRNPs and the subsequent appearance of snRNP-containing clusters of interchromatin granules, but not coiled body formation. These data demonstrate that snRNP associations with coiled bodies and interchromatin granules are differentially regulated during the cell division cycle and suggest that these structures play distinct roles connected with snRNP structure, transport, and/or function.

Biological Transport↗

PubMed1999

Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

Cell Line↗

PubMed1999

Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.

Anion Exchange Resins↗

PubMed1999

Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems.

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

Amino Acid Motifs↗