David Frendewey

Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing

ABSTRACT Myb-related cdc5p is required for G2/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiaesplicing factor Prp19p. cwf1+ is allelic to theprp5 + gene defined by the S. pombesplicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 andprp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lackingcdc5 + function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.

Cell-division-cycle defects associated with fission yeast pre-mRNA splicing mutants.

Abstract We have isolated six new pre-mRNA splicing mutants (prp) from a collection of temperature-sensitive (ts–) Schizosaccharomyces pombe strains. The prp mutants are defective in the splicing of both messenger RNA and U6 small nuclear RNA precursors. A single recessive mutation is responsible for both the ts– growth and the splicing phenotypes in each of the prp mutants. The six prp mutations are unlinked and fall into separate complementation groups. Two are allelic with the previously described prp3 and prp4 mutations; the remaining four define the new alleles prp5-1, prp6-1, prp7-1, and prp9-1. The six mutants exhibit three splicing phenotypes: accumulation of unspliced precursor at the restrictive but not at the permissive temperature; accumulation of unspliced precursor at both the permissive and restrictive temperatures; and accumulation of unspliced precursor, the intron-exon lariat intermediate, and the intron lariat final product. In addition to their aberrant splicing phenotypes, the prp5-1 and prp6-1 mutants express classical cell-division-cycle defects, while prp7-1 exhibits an unusual hyphal morphology. These results suggest a connection between pre-mRNA splicing and the control of cell division in fission yeast.

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.

Producing fully ES cell-derived mice from eight-cell stage embryo injections.

In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT). To skip the chimera stage, we developed the VelociMouse method, in which injection of genetically modified ES cells into eight-cell embryos followed by maturation to the blastocyst stage and transfer to a surrogate mother produces F0 generation mice that are fully derived from the injected ES cells and exhibit a 100% GLT efficiency. The method is simple and flexible. Both male and female ES cells can be introduced into the eight-cell embryo by any method of injection or aggregation and using all ES cell and host embryo combinations from inbred, hybrid, and outbred genetic backgrounds. The VelociMouse method provides several unique opportunities for shortening project timelines and reducing mouse husbandry costs. First, as VelociMice exhibit 100% GLT, there is no need to test cross chimeras to establish GLT. Second, because the VelociMouse method permits efficient production of ES cell-derived mice from female ES cells, XO ES cell subclones, identified by screening for spontaneous loss of the Y chromosome, can be used to generate F0 females that can be bred with isogenic F0 males derived from the original targeted ES cell clone to obtain homozygous mutant mice in the F1 generation. Third, as VelociMice are genetically identical to the ES cells from which they were derived, the VelociMouse method opens up myriad possibilities for creating mice with complex genotypes in a defined genetic background directly from engineered ES cells without the need for inefficient and lengthy breeding schemes. Examples include creation of F0 knockout mice from ES cells carrying a homozygous null mutation, and creation of a mouse with a tissue-specific gene inactivation by combining null and floxed conditional alleles for the target gene with a transgenic Cre recombinase allele controlled by a tissue-specific promoter. VelociMice with the combinatorial alleles are ready for immediate phenotypic studies, which greatly accelerates gene function assignment and the creation of valuable models of human disease.

A mutation in a single gene of Schizosaccharomyces pombe affects the expression of several snRNAs and causes defects in RNA processing.

A bank of temperature sensitive (ts‐) mutants of Schizosaccharomyces pombe was screened for snRNA expression mutants using an oligodeoxynucleotide that recognizes U2 RNA. One mutant with a novel phenotype was identified that has reduced steady‐state levels of the spliceosomal snRNAs U1, U2, U4, U5 and U6. In addition, the mutant exhibits a temperature‐dependent accumulation of aberrant U2 and U4 transcripts elongated at their 3′ end. The steady‐state concentration of the RNA component of RNase P is also reduced in the mutant, whereas the amount of U3 RNA, 7SL RNA, tRNA, rRNA and mRNA are the same as wild‐type. Pre‐mRNA, pre‐tRNA and U6 RNA precursor processing are impaired in the mutant. Genetic analysis demonstrates that the snRNA defects are tightly linked to the ts‐ growth defect and are recessive. We have named this mutant snm1 to indicate a defect in snRNA maintenance. The data on snm1 suggest that a single trans‐acting factor is essential for the maintenance of steady‐state levels of several snRNAs and for proper 3′ end formation of U2 and U4 RNAs.

Use of RNase H and primer extension to analyze RNA splicing

A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5 splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3 splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo.

Pac1 ribonuclease of Schizosaccharomyces pombe.

Publisher Summary The Pac1 RNase of the fission yeast Schizosaccharomyces pombe is a doublestrand-specific endoribonuclease (dsRNase) whose structure, biochemical properties, and biological functions place it in the RNase III family. This chapter describes the methods that have been used to define the biological functions and biochemical properties of the Pac1 RNase. The pac1 + gene issolated as a multicopy suppressor of snm1, a temperature-sensitive (ts) mutant that maintains reduced steady state amounts of the spliceosomal small nuclear RNAs (snRNAs) and accumulates Y-extended snRNA transcripts. Subsequent work showed that the snml mutation lies in the pac1 + gene and established the requirement for the Pac1 RNase in the 3′ processing of the pre-U2 RNA and presumably other snRNA precursors. Mutant Pac1 alleles, such as snm1, also accumulate Y-extended pre-rRNA, and purified Pac1 RNase cleaves a pre-rRNA at in vivo RNA processing sites. These results established a role for Pac1 in rRNA synthesis, a function shared by all members of the RNase III family.