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PubMed2006

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions.

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

Affinity Labels↗

PubMed2001

A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly.

We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.

Amino Acid Sequence↗

PubMed2004

Definition of a spliceosome interaction domain in yeast Prp2 ATPase.

The Saccharomyces cerevisiae splicing factor Prp2 is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43. We examined the role of each domain of Prp2 by deletion mutagenesis. Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity. Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2(Ts) strain, suggesting that the C-domain interferes with the activity of the Prp2(Ts) protein. A genetic approach was then taken to study interactions between Prp2 and the spliceosome. Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed. We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function. Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding. This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.

Amino Acid Sequence↗

PubMed2004

New tertiary constraints between the RNA components of active yeast spliceosomes: a photo-crosslinking study.

Elucidation of the three-dimensional (3D) structures of the two sequential active sites in spliceosomes is essential for understanding the mechanism of premessenger RNA splicing. The mechanism is predicted to be catalyzed by the small nuclear RNA (snRNA) components of spliceosomes. To obtain new tertiary constraints between the RNA components, we produced and mapped crosslinks between U6 snRNA and the proximal RNAs of active yeast spliceosomes ("yeast" in this report is Saccharomyces cerevisiae). Thus, specific sites in U6, when substituted with a photoreactive 4-thiouridine or 5-iodouridine, produced spliceosome-dependent crosslinks to U2 snRNA, or in one case, to the pre-mRNA substrate. One set of U2-U6 crosslinks formed before the Prp2p-dependent step of spliceosome assembly, whereas another set formed during or after this step but before the first chemical step of splicing. This latter set of crosslinks formed across U2-U6 helix I. Importantly, this set provides new tertiary constraints for developing 3D models of fully assembled yeast spliceosomes, which are poised for the first chemical step of splicing.

Adenosine Triphosphate↗

PubMed2002

Anti-sm autoantibodies in systemic lupus target highly basic surface structures of complexed spliceosomal autoantigens.

Autoantibodies directed against spliceosomal proteins are a common and specific feature of systemic lupus erythematosus. These autoantibodies target a collection of proteins, including Sm B, B', D1, D2, and D3. We define the common antigenic targets of Sm D2 and D3 and examine their role in spliceosomal autoimmunity. Our results define nine major common epitopes, five on Sm D2 and four on Sm D3. These epitopes have significantly higher (more basic) isoelectric points than do nonantigenic regions. In fact, this association is of sufficient power to make isoelectric point an excellent predictor of spliceosomal antigenicity. The crystallographic structure of Sm D2 and D3 is now partially described. The anti-Sm D2 and D3 antigenic targets are located on the surface of the respective three-dimensional complexed proteins, thereby suggesting that these epitopes are accessible in the native configuration. All but one of these nine epitopes conspicuously avoid the specific regions involved in intermolecular interactions within the spliceosomal complex. One of the D3 epitopes (RGRGRGMGR) has significant sequence homology with a major antigenic region of Sm D1 (containing a carboxyl-terminal glycine-arginine repeat), and anti-D3 Abs cross-react with this epitope of Sm D1. These results demonstrate that spliceosomal targets of autoimmunity are accessible on native structure surfaces and that cross-reactive epitopes, as well as structural associations of various spliceosomal Ags, may be involved in the induction of autoimmunity in systemic lupus.

Adolescent↗

PubMed1995

The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing.

Pre-mRNA processing occurs by assembly of splicing factors on the substrate to form the spliceosome followed by two consecutive RNA cleavage-ligation reactions. The Prp2 protein hydrolyzes ATP and is required for the first reaction (Yean SL, Lin RJ, 1991, Mol Cell Biol 11:5571-5577; Kim SH, Smith J, Claude A, Lin RJ, 1992, EMBO J 11:2319-2326). The Saccharomyces cerevisiae SPP2 gene was previously identified as a high-copy suppressor of temperature-sensitive prp2 mutants (Last RL, Maddock JR, Woolford JL Jr, 1987, Genetics 117:619-631). We have characterized the function of Spp2p in vivo and in vitro. Spp2p is an essential protein required for the first RNA cleavage reaction in vivo. Depletion of Spp2p from yeast cells results in accumulation of unspliced pre-mRNAs. A temperature-sensitive spp2-1 mutant accumulates pre-mRNAs in vivo and is unable to undergo the first splicing reaction in vitro. However, spliceosomal complexes are assembled in extracts prepared from the mutant. We show that Spp2p function is required after spliceosome assembly but prior to the first reaction. Spp2p associates with the spliceosome before the first RNA cleavage reaction and is likely to be released from the spliceosome following ATP hydrolysis by Prp2p. The Prp2 and Spp2 proteins are capable of physically interacting with each other. These results suggest that Spp2p interacts with Prp2p in the spliceosome prior to the first cleavage-ligation reaction. Spp2p is the first protein that has been found to interact with a DEAD/H box splicing factor.

Adenosine Triphosphate↗

PubMed1996

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

Animals↗

PubMed1996

Analysis of small nuclear RNAs in a precatalytic spliceosome.

U1 small nuclear RNA plays an important role in early stages of intron recognition and spliceosome assembly. The 5' splice site of the premessenger RNA base-pairs with the 5' end of U1; however, that interaction appears to be replaced by U5 and U6 at later stages of the splicing process. It has not been established when this transition occurs nor what factors are required for the transition. The PRP2 gene of Saccharomyces cerevisiae encodes an RNA-dependent ATPase that belongs to the DEAH putative RNA helicase family. A spliceosome can be assembled in the absence of PRP2 but the ATPase activity of PRP2 is required for the onset of the catalytic reactions in the spliceosome. By probing the precatalytic spliceosome formed in temperature-sensitive prp2 mutant extracts with oligonucleotides complementary to snRNAs, we found that the 5' end of U1 was sensitive to RNase H digestion whereas the 5' splice site-interacting region of U6 became resistant. Furthermore, by treating with heparin, a spliceosome depleted of U1 snRNA was isolated that subsequently underwent splicing with additional protein factors and ATP. Thus, these results indicate that PRP2 is not responsible for the transition from U1 to U6 in the spliceosome and that the entire U1 snRNA is not involved in the catalytic phase of pre-mRNA splicing.

Adenosine Triphosphatases↗

PubMed1997

Inhibition of mammalian spliceosome assembly and pre-mRNA splicing by peptide inhibitors of protein kinases.

Four peptides are shown to block mammalian spliceosome assembly and pre-mRNA splicing in vitro. Previously, these peptides have been shown to inhibit Ca2+-dependent calmodulin kinase II (CaMK II) via distinct mechanisms. One is a competitive inhibitor of the kinase, two interfere with autophosphorylation events, and one competes for binding to calmodulin, a CaMK II-activating protein. However, because EGTA does not inhibit splicing, the involvement of CaMK II itself in splicing is unlikely; rather, a protein similar to CaMK II may be involved in spliceosome assembly and splicing. Two of the inhibitory peptides, the calmodulin binding domain (CBD) and glycogen synthase (GS) fragment, block assembly of spliceosomal complex C. These peptides inhibited splicing if they were added to reactions any time within the first 10 min of splicing assays. No inhibition of spliceosome assembly or splicing occurred in the presence of randomized versions of the CBD or GS peptide. Additionally, the GS peptide inhibited splicing when added to assays at later time points, despite the fact that spliceosomal complex C had formed. Cumulatively, these analyses suggest that the peptides inhibit at least two distinct events in the spliceosomal cycle. The first event occurs early during in vitro splicing. For this event, prolonged incubations of splicing reactions do not result in a recovery of splicing activity. The second event occurs later and represents a slowing of an essential step, because splicing activity can be recovered in prolonged incubations. Peptides known to inhibit protein kinase A and protein kinase C had no effect on pre-mRNA splicing, underscoring the specificity of the observed inhibitory effects.

Amino Acid Sequence↗

PubMed1988

Electron microscopic identification of the yeast spliceosome.

We have partially purified the yeast spliceosome by differential sedimentation in glycerol gradients. By electron microscopy we have identified a particle in these fractions that is the spliceosome. In 100 mM KCl buffer, the yeast spliceosome is an ovoid disc with the dimensions of 20 x 23.5 nm with a central indentation. To verify that these ovoid particles were spliceosomes, specific labels were used to tag them. These tagged spliceosomes were then identified in the electron microscope. The salt dependent shift of sedimentation rate for the spliceosome can be explained by a change in size of the particle.

Bacterial Proteins↗

PubMed1994

Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism.

Splicing of mRNA precursors (pre-mRNA) is preceded by assembly of the pre-mRNA with small nuclear ribonucleoprotein particles (snRNPs) and protein factors to form a splicesome. Here we show that stimulating Ser/Thr-specific protein dephosphorylation selectively inhibits an early step during mammalian spliceosome assembly. Treatment of HeLa nuclear splicing extracts with human protein phosphatase 1 (PP1) expressed in Escherichia coli, or PP1 purified from rabbit skeletal muscle, prevents pre-spliceosome E complex (early complex) formation and stable binding of U2 and U4/U6.U5 snRNPs to the pre-mRNA. PP1 does not inhibit splicing catalysis if added after spliceosome assembly has taken place. Addition of purified SR protein splicing factors restores spliceosome formation and splicing to PP1-inhibited extracts, consistent with SR proteins being targets regulated by phosphorylation. These data extend earlier observations showing that splicing catalysis, but not spliceosome assembly, is blocked by inhibiting protein phosphatases. It therefore appears that pre-mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation.

Adenosine Triphosphate↗

PubMed1998

A limited lupus anti-spliceosomal response targets a cross-reactive, proline-rich motif.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of large amounts of characteristic autoantibodies. Anti-Sm and anti-nRNP (also known as anti-spliceosome) autoantibodies are among these. Previous epitope mapping of anti-spliceosomal antibodies has identified multiple antigenic determinants within this complex immune response. In this report we describe an SLE patient with a relatively simple, long-lasting anti-spliceosomal response. In the earliest serum sample tested the autoimmune response appeared restricted to the similar peptides PPPGMR(P,G)P of Sm B/B', PAPGMRPP of nRNP C, and PPPGMIPP of nRNP A. Unlike all the other tested lupus patients with anti-spliceosomal autoantibodies, in this patient these proline-rich epitopes remained the primary target for humoral autoimmunity in subsequent serum samples collected over many years. Absorption of anti-PAPGMRPP antibodies also removed binding to PPPGMR(P,G)P and PPPGMIPP. Isolated antibodies to PAPGMRPP were capable of binding Sm B/B', nRNP C and nRNP A by Western blot. For this particular SLE patient the autoimmune response observed against these three proteins is a single cross-reactive response against a similar proline-rich motif. Also, this patient has failed to undergo the B cell epitope spreading, typically observed in the naturally arising autoimmune response against the spliceosome in human lupus patients.

Adult↗

PubMed2004

Three-dimensional structure of the native spliceosome by cryo-electron microscopy.

Splicing of pre-mRNA occurs in a multicomponent macromolecular machine--the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins on exogenously added pre-mRNA. In contrast, splicing in vivo occurs in preformed particles where endogenous pre-mRNAs are packaged with all five spliceosomal U snRNPs (penta-snRNP) together with other splicing factors. Here we present a three-dimensional image reconstruction by cryo-electron microscopy of native spliceosomes, derived from cell nuclei, at a resolution of 20 angstroms. The structure revealed an elongated globular particle made up of two distinct subunits connected to each other leaving a tunnel in between. We show here that the larger subunit is a suitable candidate to accommodate the penta-snRNP, and that the tunnel could accommodate the pre-mRNA component of the spliceosome. The features this structure reveals provide new insight into the global architecture of the native splicing machine.

Cell Nucleus↗

PubMed2005

Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex.

Coupling between transcription and pre-mRNA splicing is a key regulatory mechanism in gene expression. Here, we investigate cotranscriptional spliceosome assembly in yeast, using in vivo crosslinking to determine the distribution of spliceosome components along intron-containing genes. Accumulation of the U1, U2, and U5 small nuclear ribonucleoprotein particles (snRNPs) and the 3' splice site binding factors Mud2p and BBP was detected in patterns indicative of progressive and complete spliceosome assembly; recruitment of the nineteen complex (NTC) component Prp19p suggests that splicing catalysis is also cotranscriptional. The separate dynamics of the U1, U2, and U5 snRNPs are consistent with stepwise recruitment of individual snRNPs rather than a preformed "penta-snRNP", as recently proposed. Finally, we show that the cap binding complex (CBC) is necessary, but not sufficient, for cotranscriptional spliceosome assembly. Thus, the demonstration of an essential link between CBC and spliceosome assembly in vivo indicates that 5' end capping couples pre-mRNA splicing to transcription.

Binding Sites↗

PubMed1997

The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis.

Spinal muscular atrophy (SMA) is an often fatal neuromuscular disease that has been directly linked to the protein product of the Survival of Motor Neurons (SMN) gene. The SMN protein is tightly associated with a novel protein, SIP1, and together they form a complex with several spliceosomal snRNP proteins. Here we show that the SMN-SIP1 complex is associated with spliceosomal snRNAs U1 and U5 in the cytoplasm of Xenopus oocytes. Antibodies directed against the SMN-SIP1 complex strongly interfere with the cytoplasmic assembly of the common (Sm) snRNP proteins with spliceosomal snRNAs and with the import of the snRNP complex into the nucleus. Thus, the SMN-SIP1 complex is directly involved in the biogenesis of spliceosomal snRNPs. Defects in spliceosomal snRNP biogenesis may, therefore, be the cause of SMA.

Antibodies, Monoclonal↗

PubMed2002

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Protein Biosynthesis↗

PubMed1999

Resolution of the mammalian E complex and the ATP-dependent spliceosomal complexes on native agarose mini-gels.

A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.

Adenosine Triphosphate↗

PubMed2004

Three-dimensional structure of C complex spliceosomes by electron microscopy.

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 A. The structure reveals a particle of dimensions 27 x 22 x 24 nm with a relatively open arrangement of three primary domains.

Cryoelectron Microscopy↗