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O’Keefe and Beggs, Page 1 Summary: Genetic analysis in yeast by plasmid shuffle O’Keefe and Beggs, Page 2 Title: Yeast genetics to investigate the function of core pre-mRNA splicing factors Raymond T. O’Keefe1 and Jean D. Beggs2 1Faculty 2Wellcome of Life Sciences, The University of Manchester, Manchester, M13 9PT, United Kingdom Trust Centre for Cell Biology, University of Edinburgh, King’s Buildings, Edinburgh, EH9 3JR, United Kingdom. *Address correspondence to: Raymond T. O’Keefe, Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, Fax 44 (0)161 275 5082, E-mail: [email protected] 1. Abstract The yeast Saccharomyces cerevisiae has been used extensively to investigate functions of RNA and protein components of the spliceosome. As many of the yeast core splicing factors are evolutionarily conserved with human counterparts, information gained in yeast can be directly applied to humans. The ease with which budding yeast can be manipulated genetically has resulted in numerous resources that make genetic analysis in this organism routine. The function of any gene that codes for a spliceosomal component can be analyzed in yeast and its genetic interactions investigated using genetic screens. Synthetic enhancement or suppression occur when a mutation in one gene respectively exacerbates or alleviates the severity of a mutation in another gene, indicating a functional interaction. In the case of splicing factors this can lead to new information on the mechanisms of splicing. This chapter describes methods for functional analysis and synthetic enhancement/lethality screens in yeast. Keywords: Saccharomyces cerevisiae, synthetic enhancement, synthetic lethality O’Keefe and Beggs, Page 3 2. Theoretical background 2.1 The use of yeast Saccharomyces cerevisiae as a model system for studying spliceosomal factors The spliceosome is composed of over a hundred protein factors and five small nuclear RNAs (snRNAs) (see chapters by Lührmann and Rymond). Compositions of both human and yeast core spliceosomes are now known, and many protein factors and all the snRNAs are evolutionarily conserved. Yeast have been used to investigate mutations in core spliceosome factors known to cause disease in humans [1], and are being used to dissect the network of genetic interactions between different core spliceosomal components [2]. Overall, using yeast has complemented work in humans and has provided key information on spliceosome function. 2.2 Yeast genetics S. cerevisiae can be maintained in diploid and haploid states and induced from one to the other easily. Yeast/bacterial shuttle plasmids are available with marker genes such as URA3, TRP1, HIS3 and LEU2, suitable for selection in auxotrophic yeast strains. The pRS series of plasmids is one of the most versatile [3]. Yeast strains are available with these marker genes deleted, including the BY strain used in the large scale yeast deletion project [4]. Transformation of yeast and homologous recombination between added DNA and endogenous chromosomes are both efficient processes making genetic manipulation straight forward. Since sequencing of the yeast genome many resources have been assembled to investigate yeast gene function including deletion of most of the known and predicted protein coding genes [5]. These “knockout” strains are useful for functional and genetic analysis of genes. Deletions in essential genes are maintained in diploid strains that can be complemented with a plasmid-encoded copy then sporulated to produce a haploid strain. A URA3-containing plasmid is usually the complementing plasmid as it allows positive and O’Keefe and Beggs, Page 4 negative selection with plates lacking uracil or containing 5-fluoro orotic acid (5-FOA) respectively [6]. By using negative selection of a URA3 plasmid functional analysis of essential genes can be investigated by plasmid shuffle where a haploid knockout strain complemented with a URA3-containing wild-type plasmid is transformed with a second plasmid with a different marker and carrying a mutant allele. Growth on 5-FOA selects for cells that lose the URA3 plasmid and mutant phenotype can be tested. LYS2 is another counter selectable marker and ADE2/ADE3 act as visual markers through red/white colony colour. See [7] for more background on yeast genetics, recipes for yeast medium/plates and other yeast methods. 2.3 Synthetic genetic interactions Availability of deletions in non-essential genes and conditional mutants in essential genes has allowed investigation of genetic interactions between core splicing machinery. Synthetic enhancement/lethality occurs when one gene mutation exacerbates the severity of another gene mutation. Alternatively, a second mutation may suppress the phenotype of the first mutation. Traditionally this type of analysis was used to uncover genetic interactions between different genes where the pathway or complex was uncharacterized. More recently, with molecular complexes like the spliceosome where the purified complex has been identified by mass spectrometry, more directed analyses can be employed to determine genetic interaction networks between core spliceosomal factors. This analysis can provide important insights into mechanistic aspects of core factors. There are many examples of synthetic lethality between genes encoding splicing factors, e.g. this approach identified an extensive genetic interaction network between SNU114 and spliceosomal snRNAs that suggests how snRNA interactions may be sensed by Snu114p [2]. In a highly informative example of suppression, requirement for Prp28p, an essential DExH/D-box splicing factor, was suppressed by mutations in U1 O’Keefe and Beggs, Page 5 snRNP protein Yhc1p, providing support for Prp28p in destabilising U1 snRNP at the 5’splice site [8]. Furthermore, effects of various prp8 mutant alleles in synthetic enhancement or suppression of mutations in other splicing factors that affect the first or second catalytic step of splicing led to the proposal that first and second step spliceosome conformations are in equilibrium [9,10]. High through-put genome-wide synthetic genetic screens can be performed, in which a mutation of interest is combined with the >4,000 deletions of non-essential yeast genes or with conditional alleles of essential genes [11]. Such approaches have aided in characterisation of core spliceosomal proteins of unknown function [12,13], however, these large scale screens require access to high through-put robotic equipment. We describe here methods to perform genetic screens between pairs of genes in any laboratory. 3. Protocol 3.1 Plasmid shuffle for functional analysis and production of conditional mutations in essential yeast genes The basis of plasmid shuffle is construction of a haploid yeast strain where a gene of interest is deleted and complemented by a wild-type gene copy on URA3 plasmid pRS416 (Figure 1). This strain is then transformed with another pRS plasmid (usually with HIS3 or LEU2 marker) containing a mutant allele of the gene to test functionally (Figure 1). This strain is grown on 5FOA to counter select the URA3 plasmid and growth phenotype of the mutation is assessed (Figure 1). 3.1.1 Construction of complementing URA3 plasmid and a plasmid for mutagenesis Design PCR primers to amplify 500 nucleotides upstream and downstream of open reading frame of gene. This is usually sufficient to complement a gene knockout. PCR primers should O’Keefe and Beggs, Page 6 also contain at their 5’ ends recognition sequences for restriction enzymes compatible with PCR product cloning into multi-cloning site (MCS) of pRS416 plasmid. PCR amplify yeast genomic DNA using a high fidelity polymerase following manufacturers instructions. An aliquot of PCR product is checked by agarose gel electrophoresis and, if of predicted size, purify with commercially available PCR cleanup kit. Digest PCR product with appropriate restriction enzymes, purify and ligate into a similarly cut pRS416 plasmid. Candidate colonies are cultured and plasmid isolated by miniprep then sequenced to identify correct plasmid. The gene is then transferred to a HIS3 or LEU2 plasmid for mutagenesis. 3.1.2 Transformation of diploid knockout strain Diploid knockout strains are available from Open Biosystems or EUROSCARF with KanMX4 cassette gene replacement which is selected for by growth on Geneticin (G418). These strains are transformed with a complementing pRS416 plasmid using an established method [14]. Protocol 1: Transformation of yeast cells Use diploid knockout strain freshly streaked on YPD-G418 for single colonies. 1. Inoculate single colony into 5ml YPD broth in 15 ml sterile tube and incubate 16-20hrs with shaking at 300C. 2. Dilute cells in 25ml YPD broth in 50 ml sterile tube to A600 of 0.5units 3. Incubate with shaking at 300C until A600 = 2 (approximately 4 hrs.) 4. Pellet cells at 2000 g for 5 minutes then decant supernate. 5. Resuspend pelleted cells in 25ml sterile water then pellet cells at 2000 g for 5 minutes and decant supernate. 6. Resuspend cells in 700µl sterile 0.1M lithium acetate and transfer to a sterile 1.5ml microcentrifuge tube. 7. Pellet cells 10,000 g for 10 seconds then aspirate supernate. 8. Resuspend cells in 200µl 0.1M lithium acetate. O’Keefe and Beggs, Page 7 9. For each transformation aliquot 50µl of cells to a new sterile 1.5ml microcentrifuge tube. 10. Pellet cells 10,000 g 15 seconds then aspirate supernate. 11. Add to pelleted cells the following: 240µl 50% polyethylene glycol 3500 36µl 1M lithium acetate 50µl 2mg/ml salmon sperm DNA (boiled for 5 mins, then on ice) 0.2-1.0µg DNA in a volume of 34µl 12. Vortex to resuspend cells. 13. Incubate at 300C for 30 minutes. 14. Incubate at 420C for 30 minutes. 15. Pellet cells 2000 g for 15 seconds. 16. Gently resuspend cells in 1ml sterile water and gently spread 50-200µl aliquots on the appropriate selective plate (SD-Ura). 17. Grow cells at 300C for 3 days 3.1.4 Yeast sporulation and tetrad dissection Transform diploid knockout strain with complementing pRS416 URA3 plasmid, sporulate then identify haploid knocked out for the gene of interest and containing complementing plasmid. Sporulation Single colonies from transformation are resuspended in 20µl of sterile water and dribbled down separately onto a sporulation plate. Plates are left at room temperature for 5 – 10 days to allow sporulation to occur. Tetrad dissection Resuspend sporulated cells in 10µl sterile water. Mix with equal volume of lyticase (2.5mg/ml) and incubate at room temperature for 5-15 minutes to digest cell wall. Dribble mixture down side of YPD plate and identify spore tetrads using a light microscope. Separate four spores of several tetrads on YPD plate. Incubate plate 30ºC for 3 days. If URA3 plasmid with wild-type O’Keefe and Beggs, Page 8 gene complements the gene knockout then three or four viable spores should be recovered. Transfer spores to different plates to test for markers. Haploid knockout strains with complementing URA3 plasmid should grow on G418 YPD and SD–Ura. If the original diploid strain was knocked out for HIS3 and LEU2 then you would expect no growth on SD–His and/or SD–Leu. The haploid strains identified should be tested for growth on 5-FOA plates. A knockout strain should not grow on 5-FOA whereas a non-knockout will. The identified knockout strain with complementing URA3 plasmid can be used for functional analysis of the essential gene or for construction of a double knockout strain for synthetic enhancement/lethality analysis. 3.1.5 Functional analysis of essential yeast genes by plasmid shuffle Haploid knockout strain with complementing URA3 plasmid identified above is transformed (Protocol 1) with a HIS3 pRS plasmid with wild-type gene as positive control, HIS3 pRS plasmid without wild-type gene as negative control or HIS3 pRS plasmid with mutation in gene of interest. Single colonies obtained from transformations are streaked on 5-FOA plates in triplicate and incubated at 16ºC, 30ºC and 37ºC with a positive and negative control on the same plate. This method can provide viable mutants for the synthetic enhancement/lethal analysis below. 3.2 Genetic interaction analysis By combining non-lethal mutations in two essential genes one can determine whether they interact genetically. This analysis can be carried out by plasmid shuffle with a haploid double knockout strain where two knockouts are complemented by a single URA3 plasmid containing both wild-type genes. Transformation of this strain with two non-lethal mutants on separate plasmids with different markers (HIS3 and LEU2), then growth on 5-FOA, will determine whether there is synthetic enhancement/lethality between the two genes (Figure 2). If one of O’Keefe and Beggs, Page 9 the mutations causes a growth phenotype by itself, such as cold- or heat-sensitivity, suppression of the defect by the second mutation is also possible. 3.2.1 Construction of double gene URA3 plasmid A URA3 marked plasmid containing two wild-type genes for complementation of the two gene deletions can be constructed as described above for a URA3 plasmid containing a single gene (3.1.1) by inserting two genes into the MCS instead of one. 3.2.2 Construction of double knockout strains The construction of a double knockout strain with a complementing URA3 plasmid with the two wild-type genes can be carried out by two different methods. The first method involves mating two haploid knockout strains of opposite mating types where each knockout is complemented by a single URA3 plasmid. Mating can be simply induced by mixing the two opposite mating types on a YPD plate. Following mating the resulting diploids are grown on 5FOA to cure them of the single URA3 plasmids. The heterozygous double knockout diploid (containing one wild-type and one deleted allele of each gene) is then transformed (Protocol 1) with another URA3 plasmid with the two wild-type genes for each knockout. The resulting strain is then sporulated and tetrads dissected (3.1.4). Haploid spores are then identified that contain the double knockout with a complementing URA3 plasmid with both wild-type genes. This strain will be the basis for the synthetic enhancement/lethality experiments. The second method for the construction of a double knockout strain with a complementing URA3 plasmid would be to start with a heterozygous or homozygous single knockout diploid strain and delete the second gene by homologous recombination with a knockout cassette that has a different selectable marker such as hphNT1 which provides resistance to hygromycin [15]. The double knockout diploid is then transformed (Protocol 1) with another URA3 plasmid with the two wild-type genes for each knockout. The resulting strain is then sporulated and tetrads dissected (3.1.4). Haploid spores are then identified that contain the double knockout with a O’Keefe and Beggs, Page 10 complementing URA3 plasmid with both wild-type genes. This strain will be the basis for synthetic enhancement/lethality experiments. 3.2.3 Genetic screens Synthetic enhancement/lethal screens use a haploid double knockout strain with complementing URA3 plasmid encoding both wild-type genes, transformed (Protocol 1) with plasmids with markers like HIS3 and LEU2 and containing mutations in each gene. One control transformation should be a negative control of a HIS3 and LEU2 plasmid without either test gene. Another control transformation should be a positive control of a HIS3 and LEU2 plasmid with wild-type copies of both genes. Single colonies obtained from these transformations are streaked out on 5-FOA plates in triplicate and incubated at 16ºC, 30ºC and 37ºC. 4. Example of an experiment Figure 3 is a typical plate from a synthetic enhancement/lethality experiment. The plate includes positive and negative controls as well as an example of a synthetic lethal interaction. 5. Troubleshooting Problem No colonies obtained from transformation No cell growth on 5-FOA plates Reason + Solution Not enough DNA used in transformation, increase DNA concentration especially if transforming two plasmids Medium used to plate cells incorrect, check genotype of cells and be sure to add all appropriate supplements Plating technique too vigorous, gently spread cells onto plates following transformation Medium used to plate cells incorrect, check genotype of cells and be sure to add all appropriate supplements Too few cells streaked onto 5-FOA O’Keefe and Beggs, Page 11 Figure Legends Figure 1 – Plasmid shuffle for functional analysis of essential yeast genes Haploid knockout strain with complementing wild-type gene on URA3 plasmid is transformed with a HIS3 plasmid with mutation in gene of interest. Growth on 5-FOA then selects for cells that have lost the URA3 plasmid and the growth phenotype of the mutation is assessed. Figure 2 – Genetic interaction analysis by plasmid shuffle Haploid double knockout strain with complementing double wild-type gene URA3 plasmid is transformed with HIS3 and LEU2 plasmids with mutations in the genes of interest. Growth on 5-FOA then selects for cells that have lost the URA3 plasmid and the genetic interaction phenotype of the two mutations combined is assessed. Figure 3 – Example of the analysis 5-FOA plate showing a typical synthetic genetic analysis of mutations in two essential yeast genes. The plate displays all the controls that should be included and provides an example of a synthetic lethal interaction. Plate supplied by Rebecca Hogg. References: [1] Boon, K.L., Grainger, R.J., Ehsani, P., Barrass, J.D., Auchynnikava, T., Inglehearn, C.F. and Beggs, J.D. (2007) prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nat Struct Mol Biol 14, 1077-1083. [2] Frazer, L.N., Lovell, S.C. and O'Keefe, R.T. (2009) Analysis of Synthetic Lethality Reveals Genetic Interactions Between the GTPase Snu114p and snRNAs in the Catalytic Core of the Saccharomyces cerevisiae Spliceosome. Genetics In press. O’Keefe and Beggs, Page 12 [3] Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27. [4] Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P. and Boeke, J.D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115-132. [5] Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., LucauDanila, A., Anderson, K., Andre, B. et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387-391. [6] Boeke, J.D., LaCroute, F. and Fink, G.R. (1984) A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197, 345-346. [7] Guthrie, C. and Fink, G. (1991) Guide to yeast genetics and molecular biology. Methods Enzymol 194, 1-863. [8] Chen, J.Y., Stands, L., Staley, J.P., Jackups, R.R., Jr., Latus, L.J. and Chang, T.H. (2001) Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Mol Cell 7, 227-232. [9] Liu, L., Query, C.C. and Konarska, M.M. (2007) Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat. Struct. Mol. Biol. 14, 519-526. [10] Query, C.C. and Konarska, M.M. (2004) Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. Mol. Cell. 14, 343-354. O’Keefe and Beggs, Page 13 [11] Tong, A.H., Evangelista, M., Parsons, A.B., Xu, H., Bader, G.D., Page, N., Robinson, M., Raghibizadeh, S., Hogue, C.W., Bussey, H. et al. (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364-2368. [12] Grainger, R.J., Barrass, J.D., Jacquier, A., Rain, J.-C. and Beggs, J.D. (2009) Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome. RNA In press. [13] Khanna, M., Van Bakel, H., Tang, X., Calarco, J.A., Babak, T., Guo, G., Emili, A., Greenblatt, J.F., Hughes, T.R., Krogan, N.J. et al. (2009) A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300. RNA In press. [14] Gietz, R.D. and Woods, R.A. (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol. Biol. 313, 107-120. [15] Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., MorenoBorchart, A., Doenges, G., Schwob, E., Schiebel, E. et al. (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947-962. Abbreviations 5-FOA (5-fluoro orotic acid) multi-cloning site (MCS) small nuclear RNAs (snRNAs)