Sequence-specific affinity selection of mammalian splicing complexes.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/biotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.     

Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly.

Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.     

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.     

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.     

A novel splicing factor, Yju2, is associated with NTC and acts after Prp2 in promoting the first catalytic reaction of pre-mRNA splicing

The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.     

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.     

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.     

Inactivation of splicing factors in HeLa cells subjected to heat shock

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.     

Cyclin E Associates with Components of the Pre-mRNA Splicing Machinery in Mammalian Cells

Cyclin E-cdk2 is a critical regulator of cell cycle progression from G₁ into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B′ and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.     

Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL,the U1 snRNP and U2AF underlie male exon skipping

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANS-FILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.     

Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus

Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking. Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.     

Mechanisms of fidelity in pre-mRNA splicing

The pre-mRNA splicing machinery consists of five small nuclear RNAs (U1, U2, U4, U5 and U6) and more than fifty proteins. Over the past year, important advances have been made in understanding how these factors function to achieve fidelity in splicing. Of particular note were the discoveries that the splicing factor U2AF(35) recognizes the AG dinucleotide at the 3' splice site early in spliceosome assembly, that a DEAD-box ATPase, Prp28, triggers specific rearrangements of the spliceosome, and that the splicing factor hSlu7 functions in the fidelity of AG choice during catalytic step II of splicing.     

Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing.

We have used a combination of highly specific protein phosphatase inhibitors and purified mammalian protein phosphatases to show that at least two separate Ser/Thr protein phosphatase activities are required for pre-mRNA splicing, but not for spliceosome assembly. Okadaic acid, tautomycin, and microcystin-LR, which are potent and specific inhibitors of PP1 and PP2A, two of the four major types of Ser/Thr-specific phosphatase catalytic subunits, block both catalytic steps of the pre-mRNA splicing mechanism in HeLa nuclear extracts. Inhibition of PP2A inhibits the second step of splicing predominantly while inhibition of both PP1 and PP2A blocks both steps, indicating a differential contribution of PP1 and PP2A activities to the two separate catalytic steps of splicing. Splicing activity is restored to toxin-inhibited extracts by the addition of highly purified mammalian PP1 or PP2A. Protein phosphatase activity was not required for efficient assembly of splicing complexes containing each of the U1, U2, U4/U6 and U5 snRNPs. The data indicate that reversible protein phosphorylation may play an important role in regulating the pre-mRNA splicing mechanism.     

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.     

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.     

PP1/PP2A phosphatases are required for the second step of Pre-mRNA splicing and target specific snRNP proteins

Pre-mRNA splicing is a complex and dynamic process in which protein phosphorylation and dephosphorylation both play important roles. Although specific phosphatases, such as PP1 and PP2A, have been implicated in splicing, direct evidence for their involvement has been lacking, and their exact function(s) in this process remain unknown. In this study, we show that PP1 and certain PP2A family phosphatases play essential but redundant roles in splicing. Unexpectedly, we found that these phosphatases are required principally for the second step of the splicing reaction. Furthermore, we provide evidence that components of U2 and U5 snRNPs, specifically SAP155 and U5-116 kDa, are the key spliceosomal substrates for these phosphatases. Based on these data, we propose that dephosphorylation of U2 and U5 snRNP components by PP1/PP2A family phosphatases facilitates essential structural rearrangements in the spliceosome during the transition from the first to the second step.