Soo-Chen Cheng

Functional association of essential splicing factor(s) with PRP19 in a protein complex.

We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19‐associated complex by affinity chromatography. The isolated complex is functional for complementation for the heat‐inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins.

Both Catalytic Steps of Nuclear Pre-mRNA Splicing Are Reversible

Nuclear pre–messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions. The presence of introns in spliceosomal RNAs of certain fungi has suggested that splicing may be reversible; however, this has never been demonstrated experimentally. By using affinity-purified spliceosomes, we have shown that both catalytic steps of splicing can be efficiently reversed under appropriate conditions. These results provide considerable insight into the catalytic flexibility of the spliceosome.

DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps

The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family. The detailed mechanism of their action was not well understood until recently, when Prp2 was shown to be required for the release of U2 components SF3a and SF3b, presumably to allow the binding of Cwc25 to promote the first transesterification reaction. We show here that Cwc25 and Yju2 are released after the reaction in Prp16- and ATP-dependent manners, possibly to allow for the binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction. The binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting that Cwc25 may bind to the branch site. We also show that Prp16 has an ATP-independent role in the first catalytic step, in addition to its known role in the second step. In the absence of ATP, Prp16 stabilizes the binding of Cwc25 to the spliceosome formed with branchpoint mutated pre-mRNAs to facilitate their splicing. Our results uncovered novel functions of Prp16 in both catalytic steps, and provide mechanistic insights into splicing catalysis.

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.

PRP19: a novel spliceosomal component.

We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae. The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base. Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif. Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction. Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components.

Cwc25 Is a Novel Splicing Factor Required after Prp2 and Yju2 To Facilitate the First Catalytic Reaction

ABSTRACT Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn2+. These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5′ splice site and the branch point during the first catalytic reaction.

Dynamic Interactions of Ntr1-Ntr2 with Prp43 and with U5 Govern the Recruitment of Prp43 To Mediate Spliceosome Disassembly

ABSTRACT The Saccharomyces cerevisiae splicing factors Ntr1 (also known as Spp382) and Ntr2 form a stable complex and can further associate with DExD/H-box RNA helicase Prp43 to form a functional complex, termed the NTR complex, which catalyzes spliceosome disassembly. We show that Prp43 interacts with Ntr1-Ntr2 in a dynamic manner. The Ntr1-Ntr2 complex can also bind to the spliceosome first, before recruiting Prp43 to catalyze disassembly. Binding of Ntr1-Ntr2 or Prp43 does not require ATP, but disassembly of the spliceosome requires hydrolysis of ATP. The NTR complex also dynamically interacts with U5 snRNP. Ntr2 interacts with U5 component Brr2 and is essential for both interactions of NTR with U5 and with the spliceosome. Ntr2 alone can also bind to U5 and to the spliceosome, suggesting a role of Ntr2 in mediating the binding of NTR to the spliceosome through its interaction with U5. Our results demonstrate that dynamic interactions of NTR with U5, through the interaction of Ntr2 with Brr2, and interactions of Ntr1 and Prp43 govern the recruitment of Prp43 to the spliceosome to mediate spliceosome disassembly.

Snt309p, a Component of the Prp19p-Associated Complex That Interacts with Prp19p and Associates with the Spliceosome Simultaneously with or Immediately after Dissociation of U4 in the Same Manner as Prp19p

ABSTRACT The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y. Tarn, C.-H. Hsu, K.-T. Huang, H.-R. Chen, H.-Y. Kao, K.-R. Lee, and S.-C. Cheng, EMBO J. 13:2421–2431, 1994). To identify proteins in the Prp19p-associated complex, we have isolatedtrans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using theade2-ade3 sectoring system. A novel splicing factor, Snt309p, was identified through such a screen. Although theSNT309 gene was not essential for growth ofSaccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature. Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p. Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis. Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly. These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.

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

ABSTRACT 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.

The Interaction of Prp2 with a Defined Region of the Intron Is Required for the First Splicing Reaction

ABSTRACT In Saccharomyces cerevisiae, the 3′ splice site is not required for the first catalytic reaction of splicing. We have previously reported that at least 24 nucleotides downstream of the branch point is required for the first reaction to take place, but the precatalytic spliceosome forms efficiently on the truncated pre-mRNA with only 5 nucleotides retained downstream of the branch point. The factors that mediate this length-dependent control of the first catalytic step are not known. We show here that Prp2 can be recruited to the spliceosome without interacting with pre-mRNA when the 3′ tail is short. Prp2 interacts with the intron when the 3′ tail is extended, which results in destabilization of Prp2 and, consequently, progression of the first reaction. An RNA segment at 23 to 33 nucleotides downstream of the branch point is necessary and sufficient for the ATP-dependent action of Prp2. We also show that Prp2 directly interacts with the carboxyl-terminal fragment of Brr2 by pulldown assays. We propose that Prp2 is recruited to the spliceosome via interaction with Brr2 and is spatially positioned to interact with this specific region of the pre-mRNA, which stimulates the ATPase activity of Prp2 to promote the progression of the first catalytic step.