Second-step Factor Research Articles

Genome-wide Analysis of Pre-mRNA Splicing: INTRON FEATURES GOVERN THE REQUIREMENT FOR THE SECOND-STEP FACTOR, Prp17 IN SACCHAROMYCES CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE

Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation. The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown. Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes. We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression. Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing. This analysis enabled the identification of substrates dependent on Prp17. We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a < or =13-nucleotide spacing between their branch point nucleotide and 3 ' splice site. In vitro splicing of substrates with varying branch nucleotide to 3 ' splice site distances supports the differential Prp17 dependencies inferred from the in vivo analysis. Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast, Schizosaccharomyces pombe, where the genome contains predominantly short introns. SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution. Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes.

Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions.

Saccharomyces cerevisiae PRP17 (CDC40) encodes a second-step pre-mRNA splicing factor with a role in cell division. The functions of Prp17 in specific cell cycle transitions were examined using temperature-sensitive alleles in arrest/release experiments. We find that G(1)/S and G(2)/M transitions depend on Prp17. G(1)-synchronized prp17::LEU2 cells arrest at non-permissive temperatures as unbudded haploid cells with low levels of CLN1, CLB5 and RNR1 transcripts. This indicates a Prp17 execution point at or prior to Start. Reduced levels of alpha-tubulin protein, a mitotic spindle component, underlie the benomyl sensitivity of prp17 mutants and possibly their G(2)/M arrest. Splicing of TUB1 and TUB3 transcripts, which encode alpha-tubulin, was analyzed in prp17 and other second-step factor mutants. TUB1 splicing is inefficient in prp17, prp16 and prp22, and marginally affected in prp18, slu7-1 and psf1-1. TUB3 splicing is similarly affected. In vitro splicing with TUB3 pre-mRNA demonstrates a compromised second step in prp17::LEU2 extracts, implicating a direct role for Prp17 in its efficient splicing. Genomic replacement of an intronless TUB1 gene relieves the benomyl sensitivity of prp17 mutants; however, they remain temperature sensitive, implying multiple limiting factors for mitosis. The data suggest that integration of splicing with the cell cycle is important for G(1)/S and G(2)/M transitions.

Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae.

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.

Splicing factor slt11p and its involvement in formation of U2/U6 helix II in activation of the yeast spliceosome.

Slt11p is a new splicing factor identified on the basis of synthetic lethality with a mutation in the 5' end of U2 snRNA, a region that is involved in intermolecular U2/U6 helix II interaction. Slt11p is required for spliceosome assembly. Our genetic results suggest that Slt11p is involved in the base-pairing interaction of U2/U6 helix II in vivo. We showed that the recombinant protein binds to RNAs with some degree of structural specificity. Slt11p also anneals RNA and binds to the resulting duplexes, which contain two separated helical regions. These RNA structures are reminiscent of U2/U6 helix II, which is formed concomitantly with U4/U6 stem II, and suggest that Slt11p facilitates the cooperative formation of helix II in association with stem II in the spliceosome. We show that Slt11p and Slu7p, a second-step factor, interact with each other both in vivo and in vitro and that the binding of Slu7p to Slt11p impairs the RNA-binding activity of the latter. These results suggest that the function of Slt11p is regulated by Slu7p in the spliceosome.

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing.

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.

Cholesterol oxidase from Rhodococcus equi is likely the major factor involved in the cooperative lytic process (CAMP reaction) with Listeria monocytogenes.

The CAMP reaction between Listeria monocytogenes and Rhodococcus equi was studied by a diffusion assay. Listeria monocytogenes displayed identical cooperative haemolytic effect with supernatant cultures of R. equi or with commercial cholesterol oxidase (COX). This result, even with enzymes of different sources (commercial COX is obtained from Pseudomonas spp.) suggests that this enzyme secreted by R. equi has a crucial role in the synergistic haemolytic (CAMP) reaction with L. monocytogenes. The mechanism of the cooperative lytic process between L. monocytogenes and R. equi may represent a different and novel mechanism reaction, in which the COX may not act as a conventional second-step factor, and a reaction different to the direct interaction with the cholesterol of the erythrocyte membrane may be involved.

SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.

Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.