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PubMed2006

Solution structure of human peptidyl prolyl isomerase-like protein 1 and insights into its interaction with SKIP.

The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 x 10(-7) M for the N-terminal peptide of SKIP-(59-129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed.

Amino Acid Sequence↗

PubMed1996

Processing of pre-mRNA in polytene nuclei of Chironomus tentans salivary gland cells.

Salivary gland polytene cells in the dipteran Chironomus tentans provide exceptional experimental possibilities to analyze processing of specific pre-mRNAs in intact eukaryotic cell nuclei. Here we give a brief account of how these experimental advantages can be exploited to analyze the splicing process in vivo. In multi-intron pre-mRNAs, spliceosomes assemble and splicing is initiated cotranscriptionally for all introns. Intron excision may, however, occur mainly co- transcriptionally or mainly posttranscriptionally depending on the position of each intron in relation to the remaining transcription time and intron-specific efficiencies of excision. As measured for the U2 snRNP and an SR protein, 10-15% of the spliceosomal components are bound to pre-mRNA at active gene loci at a given moment, while the majority of the spliceosomal components are present in the nucleoplasm. A continuous redistribution of the spliceosomal components takes place in the nucleus as a result of a close coupling between transcription and spliceosomal assembly.

Animals↗

PubMed2005

snRNAs as the catalysts of pre-mRNA splicing.

The spliceosome, the gigantic molecular machine that performs pre-mRNA splicing in eukaryotes, contains over 200 different proteins and five RNA molecules. The central role played by the spliceosomal RNAs in splicing has led to the hypothesis that, like the ribosome, the spliceosome is an RNA-centric enzyme and a relic from the RNA world. Recent structural studies have provided the first glimpses of the structural features of spliceosomal RNAs, and mutational analyses in vivo and in vitro have uncovered new functional roles for a catalytically essential domain. An emerging model for the active site of group II introns, a closely related class of natural ribozymes, is likely to provide a wealth of insights on structure and function of the active site of the spliceosome.

Base Sequence↗

PubMed1999

The type 2C Ser/Thr phosphatase PP2Cgamma is a pre-mRNA splicing factor.

To identify activities involved in human pre-mRNA splicing, we have developed a procedure to separate HeLa cell nuclear extract into five complementing fractions. An activity called SCF1 was purified from one of these fractions by assaying for reconstitution of splicing in the presence of the remaining four fractions. A component of SCF1 is shown to be PP2Cgamma, a type 2C Ser/Thr phosphatase of previously unknown function. Previous work suggested that dephosphorylation of splicing factors may be important for catalysis after spliceosome assembly, although the identities of the specific phosphatases involved remain unclear. Here we show that human PP2Cgamma is physically associated with the spliceosome in vitro throughout the splicing reaction, but is first required during the early stages of spliceosome assembly for efficient formation of the A complex. The phosphatase activity is required for the splicing function of PP2Cgamma, as an active site mutant does not support spliceosome assembly. The requirement for PP2Cgamma is highly specific, as the closely related phosphatase PP2Calpha cannot substitute for PP2Cgamma. Consistent with a role in splicing, PP2Cgamma localizes to the nucleus in vivo. We conclude that at least one specific dephosphorylation event catalyzed by PP2Cgamma is required for formation of the spliceosome.

Amino Acid Sequence↗

PubMed1996

Highly diverged U4 and U6 small nuclear RNAs required for splicing rare AT-AC introns.

Removal of a rare class of metazoan precursor messenger RNA introns with AU-AC at their termini is catalyzed by a spliceosome that contains U11, U12, and U5 small nuclear ribonucleoproteins. Two previously unidentified, low-abundance human small nuclear RNAs (snRNAs), U4atac and U6atac, were characterized as associated with the AT-AC spliceosome and necessary for AT-AC intron splicing. The excision of AT-AC introns therefore requires four snRNAs not found in the major spliceosome. With the use of psoralen crosslinking, a U6atac interaction with U12 was identified that is similar to a U6-U2 helix believed to contribute to the spliceosomal active center. The conservation of only limited U6atac sequences in the neighborhood of this interaction and the potential of U6atac to base pair with the 5' splice site consensus for AT-AC introns provide support for current models of the core of the spliceosome.

Base Composition↗

PubMed1997

Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro.

The splicing reaction that removes introns from pre-messenger RNAs requires the assembly of the spliceosome on the nascent transcript, proper folding of the substrate-enzyme complex, and finally, two transesterification reactions. These stages in the splicing reaction must require careful orchestration. Here we show data that suggest that the sequential phosphorylation and dephosphorylation of SR proteins mark the transition between stages in the splicing reaction. Many data had already led to the idea that phosphorylation of SR proteins could modulate their activity, when we showed that dephosphorylation of these proteins abrogates their activity in a reaction measuring conversion of pre-spliceosomes to spliceosomes (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706). Subsequently, Xiao and Manley (1997, Genes & Dev 11:334-344) showed that phosphorylated ASF/SF2, but not mock-phosphorylated ASF/SF2, activates the splicing of HIV tat pre-mRNA in reactions challenged with excess random RNA. Here we confirm and extend these two findings. Phosphorylated ASF/SF2 efficiently complemented an SR protein-deficient HeLa S100 extract in promoting the splicing of an adenovirus-2-derived pre-messenger RNA, whereas unphosphorylated ASF/ SF2 did not. Moreover, we show that, whereas unphosphorylated ASF/SF2 inhibited splicing in HeLa nuclear extracts, phosphorylation of the ASF/SF2 reversed the inhibition and enhanced splicing. We also present data that shows that dephosphorylation of ASF/SF2 is required for the first transesterification reaction once the spliceosome has assembled. Thiophosphorylated ASF/SF2, which cannot be readily dephosphorylated, can promote spliceosome assembly, but cannot promote the first transesterification reaction. These data, together with other observations, indicate for the first time a requirement for SR protein dephosphorylation in pre-messenger RNA splicing in vitro.

Adenosine Triphosphate↗

PubMed1992

Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.

Bacterial Proteins↗

PubMed2001

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop.

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.

5' Untranslated Regions↗

PubMed1998

Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.

Base Sequence↗

PubMed2004

A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing.

During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human spliceosomes that had not yet undergone catalytic activation (designated BDeltaU1), which contained U2, U4, U5, and U6, but lacked U1 snRNA. Comparison of the protein compositions of BDeltaU1 and activated B* spliceosomes revealed that, whereas U4/U6 snRNP proteins are stably associated with BDeltaU1 spliceosomes, 35S U5-associated proteins (which are present in B*) are largely absent, suggesting that they are dispensable for complex B formation. Indeed, immunodepletion/complementation experiments demonstrated that a subset of 35S U5 proteins including Prp19, which form a stable heteromeric complex, are required prior to catalytic step 1 of splicing, but not for stable integration of U4/U6.U5 tri-snRNPs. Thus, comparison of the proteomes of spliceosomal complexes at defined stages can provide information as to which proteins function as a group at a particular step of splicing.

Carrier Proteins↗

PubMed1990

Interactions of PRP2 protein with pre-mRNA splicing complexes in Saccharomyces cerevisiae.

PRP2 protein of Saccharomyces cerevisiae is required for the pre-mRNA splicing reaction but not for the early stages of spliceosome assembly. Using anti-PRP2 antibodies we demonstrate that PRP2 protein is associated with spliceosomes prior to, and throughout step 1 of the splicing reaction. Heat-inactivated prp2 protein, by contrast, does not seem to associate with spliceosomes. By elution of electrophoretically distinct spliceosomal complexes from non-denaturing gels we identify the specific complex with which PRP2 initially interacts in the pathway of spliceosome assembly.

Antibodies↗

PubMed1992

Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing.

Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.

Adenoviridae↗

PubMed1989

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

Cell Fractionation↗

PubMed1988

RNA splicing and genes.

The splicing of long transcripts of RNA (copied from DNA in the cell nucleus) into smaller, specific mRNA (ready for export to the protein-producing machinery in the cytoplasm) is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing the 5' and 3' splice sites. This complex contains the evolutionarily highly conserved small nuclear RNAs (snRNAs) U2, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRNAs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

Animals↗

PubMed2003

Sm and U2B" proteins redistribute to different nuclear domains in dormant and proliferating onion cells.

Monoclonal antibodies against the spliceosomal proteins Sm and U2B", and against p105, a protein component of interchromatin granules, were used to investigate the nuclear distribution of the splicing factors in Allium cepa L. meristematic cells. Confocal microscopy showed that in steady-state proliferating cells, the spliceosomal components were distributed into two nuclear domains: (i) a diffuse nucleoplasmic network similar to that formed by interchromatin granules and (ii) numerous Cajal bodies. These domains were the counterpart of the perichromatin fibrils and granules, interchromatin granules and Cajal bodies observed by electron microscopy after EDTA and bismuth oxynitrate stainings. Dormant cells showed a nuclear distribution of the proteins in small Cajal bodies and numerous micro-speckles, correlated with the distribution of ribonucleoproteins (RNPs) observed by electron microscopy. The spliceosomal proteins relocated to the diffuse nucleoplasmic network and Cajal bodies when the cells were released from dormancy by water soaking and they re-started their proliferative activity. Inhibition of RNA synthesis by 5,6-dichloro-1-beta- d-ribofuranosylbenzimidazole (DRB) treatment in proliferating cells demonstrated that the micro-speckles were not the morphological expression of a transcription block. Fractionation and confocal microscopy studies showed a differential association of the splicing factors with the nuclear matrix depending not only on the protein, but also on nuclear activity. Our results suggest a reversible relocation of the spliceosomal proteins between different sub-nuclear domains in physiological conditions. We report here an unusual nuclear domain in dormant nuclei, the micro-speckles, corresponding to storage sites for RNPs, which were rapidly mobilised after water imbibition.

Autoantigens↗

PubMed2005

Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets.

Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.

Alternative Splicing↗

PubMed2004

Lsm proteins promote regeneration of pre-mRNA splicing activity.

Lsm proteins are ubiquitous, multifunctional proteins that affect the processing of most RNAs in eukaryotic cells, but their function is unknown. A complex of seven Lsm proteins, Lsm2-8, associates with the U6 small nuclear RNA (snRNA) that is a component of spliceosome complexes in which pre-mRNA splicing occurs. Spliceosomes contain five snRNAs, U1, U2, U4, U5, and U6, that are packaged as ribonucleoprotein particles (snRNPs). U4 and U6 snRNAs contain extensive sequence complementarity and interact to form U4/U6 di-snRNPs. U4/U6 di-snRNPs associate with U5 snRNPs to form U4/U6.U5 tri-snRNPs prior to spliceosome assembly. Within spliceosomes, disruption of base-paired U4/U6 heterodimer allows U6 snRNA to form part of the catalytic center. Following completion of the splicing reaction, snRNPs must be recycled for subsequent rounds of splicing, although little is known about this process. Here we present evidence that regeneration of splicing activity in vitro is dependent on Lsm proteins. RNP reconstitution experiments with exogenous U6 RNA show that Lsm proteins promote the formation of U6-containing complexes and suggest that Lsm proteins have a chaperone-like function, supporting the assembly or remodeling of RNP complexes involved in splicing. Such a function could explain the involvement of Lsm proteins in a wide variety of RNA processing pathways.

Multigene Family↗

PubMed2000

Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins.

The Prp2 protein of Saccharomyces cerevisiae 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. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.

Adenosine Triphosphatases↗