Inhibition of in vitro pre-mRNA splicing in S. cerevisiae by branched oligonucleotides.

A series of V- and Y-shaped nucleic acids, related to the splicing intermediates derived from S. cerevisiae actin pre-mRNA, were prepared. The effects of such branched nucleic acids (bNAs) on the efficiency of in vitro pre-mRNA splicing in yeast were studied. The exogenous bNAs each effect the efficiency of splicing, yet to different degrees, depending on the sugar composition and topology of the molecules. Y-shaped RNAs inhibited the formation of mRNA (i.e. RNA splicing) to the greatest extent.

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.

Protein production: feeding the crystallographers and NMR spectroscopists.

Protein purification efforts for structural genomics will focus on automation for the readily-expressed proteins, and process development for the more difficult ones, such as membrane proteins. Thousands of proteins are expected to be produced in the next few years. The purified proteins will be valuable reagents for the entire research community.

RNA polymerase II subunit Rpb9 regulates transcription elongation in vivo.

RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Deltarpb9 cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Deltarpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Deltarpb9 cells revealed only a few changes, predominantly in genes related to metabolism.

Zinc stoichiometry of yeast RNA polymerase II and characterization of mutations in the zinc-binding domain of the largest subunit.

Atomic absorption spectroscopy demonstrated that highly purified RNA polymerase II from the yeast Saccharomyces cerevisiae binds seven zinc ions. This number agrees with the number of potential zinc-binding sites among the 12 different subunits of the enzyme and with our observation that the ninth largest subunit alone is able to bind two zinc ions. The zinc-binding motif in the largest subunit of the enzyme was investigated using mutagenic analysis. Altering any one of the six conserved residues in the zinc-binding motif conferred either a lethal or conditional phenotype, and zinc blot analysis indicated that mutant forms of the domain had a 2-fold reduction in zinc affinity. Mutations in the zinc-binding domain reduced RNA polymerase II activity in cell-free extracts, even though protein blot analysis indicated that the mutant subunit was present in excess of wild-type levels. Purification of one mutant RNA polymerase revealed a subunit profile that was wild-type like with the exception of two subunits not required for core enzyme activity (Rpb4p and Rpb7p), which were missing. Core activity of the mutant enzyme was reduced 20-fold. We conclude that mutations in the zinc-binding domain can reduce core activity without altering the association of any of the subunits required for this activity.

Elongin from Saccharomyces cerevisiae.

Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

Stimulation of transcription by mutations affecting conserved regions of RNA polymerase II.

Mutations that increase the low-level transcription of the Saccharomyces cerevisiae HIS4 gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in SIT1 (also known as RPO21, RPB1, and SUA8), the gene encoding the largest subunit of RNA polymerase II (RNAPII). Here we show that sit1 substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six sit1 mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to alpha-aminatin. Four sit1 substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg2+ ions essential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with PPR2, the gene encoding transcription elongation factor IIS. Some sit1 substitutions affect the selection of transcriptional start sites at the CYC1 promoter in a manner reminiscent of that of sua8 (sua stands for suppression of upstream ATG) mutations. Together with previous findings which indicate that regions D and G are in close proximity to the 3' end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the sit1 mutations results from alteration of the RNAPII active center.

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.

Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome.

Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.

Genetic evidence for selective degradation of RNA polymerase subunits by the 20S proteasome in Saccharomyces cerevisiae.

scs32 was isolated as an extragenic suppressor of a temperature-sensitive (ts) mutation (rpo26-31) in the gene encoding Rpo26p, a subunit common to yeast nuclear RNA polymerases (RNAPs). rpo26-31 also confers inositol auxotrophy, inhibits the assembly of RNAPI and RNAPII and reduces the steady-state level of Rpo26p and the largest subunit of RNAPI (Rpo11p or A190p) and RNAPII (Rpo21p). rpo26-31p accumulated to wild-type levels in the scs32 strain; nevertheless, the amount of assembled RNAPII remained at a reduced level at high temperature. Hence, scs32 only partially suppressed the ts phenotype and was unable to suppress the Ino-phenotype of rpo26-31. SCS32 is identical to PUP3, which encodes a subunit of the yeast proteasome. scs32 was able to suppress the phenotype of other ts alleles of RPO26, all of which reduce the steady-state level of this subunit. However, scs32 was unable to suppress the ts phenotype of mutant alleles of RPO21, or result in accumulation of the unstable rpo21-4p. These observations suggest that the stability of non-functional or unassembled forms of Rpo26p and Rpo21p are regulated independently.

Rpo26p, a subunit common to yeast RNA polymerases, is essential for the assembly of RNA polymerases I and II and for the stability of the largest subunits of these enzymes.

Eukaryotic nuclear RNA polymerases (RNAPs) are composed of two large subunits and a number of small polypeptides, some of which are common among these enzymes. To understand the function of Rpo26p, one of the five subunits common to yeast RNAPs, 34 different mutations have been isolated in RP026 that cause cell death in a strain carrying a temperature-sensitive (ts) mutation in the gene (RP021) encoding the largest subunit of RNAPII. These mutant alleles were grouped into three phenotypic classes (null, ts, and neutral) on the basis of the phenotype they imposed in combination with wild-type RP021. The function of Rpo26p was addressed by biochemical analysis of the ts rpo26-31 allele. The steady-state level of rpo26-31p was reduced at high temperature; this was accompanied by a decrease in the level of at least two other subunits, the largest subunits of RNAPI (A190p) and RNAPII (Rpo21p). Pulse-chase metabolic labeling and immunoprecipitation of RNAPII showed that at high temperature, rpo26-31 did not lead to dissociation of Rpo26p from the polymerase but prevented the assembly of RNAPII. Overexpression of rpo26-31 partially suppressed the ts phenotype and led to accumulation of the mutant subunit. However, overexpression only marginally suppressed the assembly defect of RNAPII. Furthermore, A190p and Rpo21p continued to accumulate at low levels under these conditions. We suggest that Rpo26p is essential for the assembly of RNAPI and RNAPII and for the stability of the largest subunits of these enzymes.

Similar upstream regulatory elements of genes that encode the two largest subunits of RNA polymerase II in Saccharomyces cerevisiae.

We have determined the location of cis-acting elements that are important for the expression of RPO21 and RPO22, genes that encode the two largest subunits of RNA polymerase II (RNAPII) in Saccharomyces cerevisiae. A series of 5'-end deletions and nucleotide substitutions in the upstream regions of RPO21 and RPO22 were tested for their effect on the expression of lacZ fusions of these genes. Deletion of sequences from -723 to -693 in RPO21, which disrupted two Reb1p-binding sites and an Abf1p-binding site, resulted in a 10-fold decrease in expression. A T-rich region downstream of these sites was also important for expression. Deletion of sequences from -437 to -392 in the RPO22-upstream, which resulted in a 30-fold decrease in expression, indicated that the Reb1p- and Abf1p-binding sites in this region were important for RPO22 expression, as was a T-rich sequence immediately downstream of these sites. The RPO21 and RPO22 upstream regions were capable of interacting in vitro (gel-mobility-shift assays) with Reb1p and Abf1p. The similarities in the type and organization of elements in the upstream regions of RPO21 and RPO22 suggest that expression of these genes may be regulated coordinately.

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Mutations in an Abf1p binding site in the promoter of yeast RPO26 shift the transcription start sites and reduce the level of RPO26 mRNA.

A binding site for the transcription factor Abf1p was identified as an important promoter element of the gene that encodes Rpo26, a subunit common to all three yeast nuclear RNA polymerases (RNAP). Mutations in the Abf1p binding site were identified among a pool of rpo26 mutant alleles that confer synthetic lethality in combination with a temperature-sensitive mutation (rpo21-4) in the gene that encodes the largest subunit of RNAPII (Rpo21p). In the presence of the wild-type allele of RPO21 these rpo26 promoter mutations confer a cold-sensitive growth defect. Electrophoretic mobility-shift assays using purified Abf1p demonstrated that Abf1p binds to the RPO26 promoter and that the promoter mutations abolish this binding in vitro. Quantitation of the amount of RPO26 mRNA showed that mutations in the Abf1p binding site reduce the expression of RPO26 by approximately 60%. Mutations that affect Abf1p binding also result in a shift of the RPO26 transcriptional start sites to positions further upstream than normal. These results suggest that binding of the Abf1p transcription factor to the RPO26 promoter is important not only in establishing the level of transcription for this gene, but also in positioning the initiation sites of transcription.

An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing.

The hydrolysis of ATP by a group of RNA-dependent ATPases (DEAD/H proteins) is required for spliceosome assembly, but not for the subsequent transesterification reactions. Little is known about the function of these ATPases in relation to the RNA conformational changes that occur in formation of active structures, in which U2/U6 small nuclear RNA (snRNA) interactions are essential for splicing to take place. Using a synthetic lethal genetic screen, we have isolated four yeast splicing factors involved in U2/U6 snRNA interactions (D.X. et al., manuscript in preparation). The RNA-dependent ATPase activity associated with one such factor, the Slt22 protein, is stimulated preferentially by annealed U2/U6 snRNAs. Both mutant slt22-1 and U2 snRNA cause a reduction in stimulation. The slt22-1 mutation blocks splicing at or before the first step, resulting in the accumulation of an unusual complex which lacks U5 snRNA. Our results indicate that the U2/U6 snRNA interactions facilitated by Slt22 are also involved in the interaction of U5 snRNA with the spliceosome.

Underproduction of the largest subunit of RNA polymerase II causes temperature sensitivity, slow growth, and inositol auxotrophy in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

Functionally redundant interactions between U2 and U6 spliceosomal snRNAs.

Base-pairing between U2 and U6 snRNAs to form intermolecular helix II has been demonstrated previously as a requirement for pre-mRNA splicing in mammalian cells. In contrast, deletion and substitution mutation experiments in yeast have indicated that helix II is not essential; instead, other regions of U2 and U6 have been proposed to pair, forming a helix called Ib. To investigate the importance of U2/U6 helices in yeast, we have systematically mutagenized the regions proposed to form helices II and Ib. Allele-specific suppression of certain U6 mutations by complementary substitutions in U2 show that helix II indeed form in yeast but that it is essential only in the presence of additional mutations that disrupt U2 stem I and the proposed helix Ib. Similarly, the proposed helix Ib is essential only when helix II is disrupted. These observations provide an explanation for apparently conflicting data in yeast and mammalian experimental systems, and identify synergistic or functionally redundant interactions between U2 and U6 snRNAs.

Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator.

The binding of TATA-binding protein (TBP) to the TATA element is the first step in the initiation of RNA polymerase II transcription from many promoters in vitro. It has been proposed that upstream activator proteins stimulate transcription by recruiting TBP to the promoter, thus facilitating the assembly of a transcription complex. However, the role of activator proteins acting at this step to stimulate transcription in vivo remains largely speculative. To test whether recruitment of TBP to the promoter is sufficient for transcriptional activation in vivo, we constructed a hybrid protein containing TBP of the yeast Saccharomyces cerevisiae fused to the DNA-binding domain of GAL4. Our results show that TBP recruited by the GAL4 DNA-binding domain to promoters bearing a GAL4-binding site can interact with the TATA element and direct high levels of transcription. This finding indicates that binding of TBP to promoters in S. cerevisiae is a major rate-limiting step accelerated by upstream activator proteins.

Mutational analysis of Saccharomyces cerevisiae U4 small nuclear RNA identifies functionally important domains.

U4 small nuclear RNA (snRNA) is essential for pre-mRNA splicing, although its role is not yet clear. On the basis of a model structure (C. Guthrie and B. Patterson, Annu. Rev. Genet. 22:387-419, 1988), the molecule can be thought of as having six domains: stem II, 5' stem-loop, stem I, central region, 3' stem-loop, and 3'-terminal region. We have carried out extensive mutagenesis of the yeast U4 snRNA gene (SNR14) and have obtained information on the effect of mutations at 105 of its 160 nucleotides. Fifteen critical residues in the U4 snRNA have been identified in four domains: stem II, the 5' stem-loop, stem I, and the 3'-terminal region. These domains have been shown previously to be insensitive to oligonucleotide-directed RNase H cleavage (Y. Xu, S. Petersen-Bjørn, and J. D. Friesen, Mol. Cell. Biol. 10:1217-1225, 1990), suggesting that they are involved in intra- or intermolecular interactions. Stem II, a region that base pairs with U6 snRNA, is the most sensitive to mutation of all U4 snRNA domains. In contrast, stem I is surprisingly insensitive to mutational change, which brings into question its role in base pairing with U6 snRNA. All mutations in the putative Sm site of U4 snRNA yield a lethal or conditional-lethal phenotype, indicating that this region is important functionally. Only two nucleotides in the 5' stem-loop are sensitive to mutation; most of this domain can tolerate point mutations or small deletions. The 3' stem-loop, while essential, is very tolerant of change. A large portion of the central domain can be removed or expanded with only minor effects on phenotype, suggesting that it has little function of its own. Analysis of conditional mutations in stem II and stem I indicates that although these single-base changes do not have a dramatic effect on U4 snRNA stability, they are defective in RNA splicing in vivo and in vitro, as well as in spliceosome assembly. These results are discussed in the context of current knowledge of the interactions involving U4 snRNA.

The central region of the yeast U4 snRNA is not important for hammerhead catalysis, but may act as a domain spacer.

Several distinct domains of U4 small nuclear (sn)RNA interact with other components of the spliceosome and are essential for pre-mRNA splicing. We have previously shown that single point mutations (Hu et al., 1995) in the central domain of the yeast U4 snRNA do not effect cell growth. In contrast to our results, this region has been reported to possess metal-binding or catalytic activity in vitro (Yang et al., 1994). In order to test if large mutations in the central domain effect cell growth and/or in vivo splicing, we have carried out further mutational analyses of this domain. A deletion of nucleotides from 62 to 88, including all those corresponding to the invariable positions of the hypothetical hammerhead, does not affect cell growth at/or above 25 degrees C. In addition, the central region (nucleotides 69-88) can be replaced by two copies of a 38-nt spacer inserted in the same orientation without any effect on cell growth or nuclear RNA splicing. These results suggest that the central domain of U4 snRNA functions primarily to separate the 5'- and 3'- domains of the snRNA by an optimal distance.