A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3- ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress

Large-scale profiling of noncoding RNA function in yeast

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs

Physiological consequences of Elongator complex inactivation in Eukaryotes

Mutations found in genes encoding human Elongator complex subunits have been linked to neurodevelopmental disorders such as familial dysautonomia (FD), rolandic epilepsy and amyotrophic lateral sclerosis. In addition, loss-of-function mutations in genes encoding Elongator complex subunits cause defects in neurodevelopment and reduced neuronal function in both mice and nematodes. The Elongator complex is a conserved protein complex comprising six subunits (Elp1p-Elp6p) found in eukaryotes. The primary function of this complex in yeast is formation of the 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side chains found on wobble uridines (U34) in tRNAs. The aim of this thesis is to investigate the physiological consequences of Elongator complex inactivation in humans and in the yeast Saccharomyces cerevisiae. Inactivation of the Elongator complex causes widespread defects in a multitude of different cellular processes in S. cerevisiae. Thus, we investigated metabolic alterations resulting from Elongator complex inactivation. We show that deletion of the S. cerevisiae ELP3 gene leads to widespread metabolic alterations. Moreover, all global metabolic alterations observed in the elp3Δ strain are not restored in the presence of elevated levels of hypomodified tRNAs that normally have the modified nucleoside mcm5s2U. Collectively, we show that modified wobble nucleosides in tRNAs are required for metabolic homeostasis. Elongator mutants display sensitivity to DNA damage agents, but the underlying mechanism explaining this sensitivity remains elusive. We demonstrate that deletion of the S. cerevisiae ELP3 gene results in post-transcriptional reduction of Ixr1p levels. Further, we show that the reduced Ixr1p levels prevent adequate Rnr1p levels upon treatment with DNA damage agents. These findings suggest that reduced Ixr1p levels could in part explain why Elongator mutants are sensitive to DNA damage agents. Depletion of Elongator complex subunits results in loss of wobble uridine modifications in plants, nematodes, mice and yeast. Therefore, we investigated whether patients with the neurodegenerative disease familial dysautonomia (FD), who have lower levels of the ELP1 protein, display reduced amounts of modified wobble uridine nucleosides. We show that tRNA isolated from brain tissue and fibroblast cell lines derived from FD patients have 64–71% of the mcm5s2U nucleoside levels observed in total tRNA from non-FD brain tissue and non-FD fibroblasts. Overall, these results suggest that the cause for the neurodegenerative nature of FD could be translation impairment caused by reduced levels of modified wobble uridine nucleosides in tRNAs. Thus, our results give new insight on the importance of modified wobble uridine nucleosides for neurodevelopment

Renting out your Condominium : A Study on Rental in the Swedish Housing Market

Detta arbete är författat på Kungliga Tekniska Högskolan i Stockholm inom kandidatprogrammet Fastighetsutveckling med fastighetsförmedling. Studien är gjord i samarbete med JM AB i Stockholm. Ägarlägenheter infördes i svensk lagstiftning år 2009. Den nya boendeformen sågs vid införandet som en möjlighet att öka valfriheten och mångfalden i boendet. Det skulle även innebära ett större inflytande över boendet, bland annat i avseende på fri uthyrning. Boendeformen har nu funnits i Sverige i över ett decennium och antalet lägenheter har successivt ökat. Idag finns lite över 3000 ägarlägenheter i Sverige. Ägarlägenheten har under lång tid varit föremål för kritik, och en av farhågorna med boendeformen är den fria uthyrningen, som framhållits kunna leda till problem på bostadsmarknaden. Det är därmed av intresse att studera hur uthyrning av ägarlägenheter tillämpas i nuläget. Syftet med denna studie är att undersöka hur uthyrning av ägarlägenheter sker i praktiken. Undersökningen görs utifrån i vilken omfattning och på vilket sätt lägenheterna hyrs ut, vilken typ av lägenheter som hyrs ut, till vem lägenheterna hyrs ut och om det finns några problem i samband med uthyrning av ägarlägenheter. Detta har gjorts genom en enkätstudie, som kompletterats med en litteraturstudie och en intervjustudie. Respondenterna till enkätstudien har tillhandahållits genom JM. I intervjustudien medverkar ordföranden för ägarlägenhetssamfälligheter, ägare av ägarlägenheter samt Hyresgästföreningen. Studien visar att majoriteten av ägare av ägarlägenheter, aldrig hyrt ut sin lägenhet. Det framgår även att de lägenheter som hyrs ut oftast är mindre lägenheter på ett till två rum och kök, och att uthyrningen oftast sker till bekantas bekanta. De flesta uthyrningar varar mellan sex månader till ett år, och uthyrningar som varar mindre än en månad är ovanliga. Nöjdhetsgraden bland uthyrare är hög. Det framgår inga omfattande problem i samband med uthyrning men av de problem som identifierats, är hantering av gemensamma utrymmen det mest återkommande.This essay was conducted at the Royal Institute of Technology in Stockholm, within the Bachelor's program of Property Development and Agency. The study was carried out in collaboration with JM AB in Stockholm. Condominiums were introduced into Swedish legislation in 2009. The new form of tenure was at the time of its introduction viewed as an opportunity to increase freedom of choice and diversity in housing as well as greater influence over one's own living, including the freedom to rent out your home. This form of tenure has existed for over a decade and has gradually increased. Today, there are just over 3000 condominiums in Sweden. Condominiums have long been the subject of criticism, and some of the concerns with this form of tenure are the opportunities for rental. This has been highlighted as a potential risk that can cause problems in the housing market. Therefore, it is of interest to study how the rental of condominiums is carried out in practice. The purpose of this study is to investigate how the rental of condominiums is carried out in practice. This is based on the extent and the way in which the apartments are rented out, what type of apartments are being rented, to whom the apartments are rented, and whether there are any problems associated with the rental of condominiums. This has been done through a survey, supplemented by a literature study and an interview study. The respondents for the survey were provided by JM. The interview study involves the chairmen of condominiums associations, owners of condominiums, and Hyresgästföreningen. The study shows that the majority of owners of condominiums have never rented out their apartment. It also appears that the apartments that are rented out are often smaller apartments with one to two rooms with a kitchen, and that the rental is most often to acquaintances of acquaintances. Most rentals last between six months to one year, and rentals that last less than a month are rare. Satisfaction among landlords is high. There are no significant problems associated with rentals, but of the problems that have been identified, the management of common spaces is the most recurring issue

Renting out your Condominium : A Study on Rental in the Swedish Housing Market

Detta arbete är författat på Kungliga Tekniska Högskolan i Stockholm inom kandidatprogrammet Fastighetsutveckling med fastighetsförmedling. Studien är gjord i samarbete med JM AB i Stockholm. Ägarlägenheter infördes i svensk lagstiftning år 2009. Den nya boendeformen sågs vid införandet som en möjlighet att öka valfriheten och mångfalden i boendet. Det skulle även innebära ett större inflytande över boendet, bland annat i avseende på fri uthyrning. Boendeformen har nu funnits i Sverige i över ett decennium och antalet lägenheter har successivt ökat. Idag finns lite över 3000 ägarlägenheter i Sverige. Ägarlägenheten har under lång tid varit föremål för kritik, och en av farhågorna med boendeformen är den fria uthyrningen, som framhållits kunna leda till problem på bostadsmarknaden. Det är därmed av intresse att studera hur uthyrning av ägarlägenheter tillämpas i nuläget. Syftet med denna studie är att undersöka hur uthyrning av ägarlägenheter sker i praktiken. Undersökningen görs utifrån i vilken omfattning och på vilket sätt lägenheterna hyrs ut, vilken typ av lägenheter som hyrs ut, till vem lägenheterna hyrs ut och om det finns några problem i samband med uthyrning av ägarlägenheter. Detta har gjorts genom en enkätstudie, som kompletterats med en litteraturstudie och en intervjustudie. Respondenterna till enkätstudien har tillhandahållits genom JM. I intervjustudien medverkar ordföranden för ägarlägenhetssamfälligheter, ägare av ägarlägenheter samt Hyresgästföreningen. Studien visar att majoriteten av ägare av ägarlägenheter, aldrig hyrt ut sin lägenhet. Det framgår även att de lägenheter som hyrs ut oftast är mindre lägenheter på ett till två rum och kök, och att uthyrningen oftast sker till bekantas bekanta. De flesta uthyrningar varar mellan sex månader till ett år, och uthyrningar som varar mindre än en månad är ovanliga. Nöjdhetsgraden bland uthyrare är hög. Det framgår inga omfattande problem i samband med uthyrning men av de problem som identifierats, är hantering av gemensamma utrymmen det mest återkommande.This essay was conducted at the Royal Institute of Technology in Stockholm, within the Bachelor's program of Property Development and Agency. The study was carried out in collaboration with JM AB in Stockholm. Condominiums were introduced into Swedish legislation in 2009. The new form of tenure was at the time of its introduction viewed as an opportunity to increase freedom of choice and diversity in housing as well as greater influence over one's own living, including the freedom to rent out your home. This form of tenure has existed for over a decade and has gradually increased. Today, there are just over 3000 condominiums in Sweden. Condominiums have long been the subject of criticism, and some of the concerns with this form of tenure are the opportunities for rental. This has been highlighted as a potential risk that can cause problems in the housing market. Therefore, it is of interest to study how the rental of condominiums is carried out in practice. The purpose of this study is to investigate how the rental of condominiums is carried out in practice. This is based on the extent and the way in which the apartments are rented out, what type of apartments are being rented, to whom the apartments are rented, and whether there are any problems associated with the rental of condominiums. This has been done through a survey, supplemented by a literature study and an interview study. The respondents for the survey were provided by JM. The interview study involves the chairmen of condominiums associations, owners of condominiums, and Hyresgästföreningen. The study shows that the majority of owners of condominiums have never rented out their apartment. It also appears that the apartments that are rented out are often smaller apartments with one to two rooms with a kitchen, and that the rental is most often to acquaintances of acquaintances. Most rentals last between six months to one year, and rentals that last less than a month are rare. Satisfaction among landlords is high. There are no significant problems associated with rentals, but of the problems that have been identified, the management of common spaces is the most recurring issue

Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile

Introduction: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm(5)) and 5-methoxycarbonylmethyl (mcm(5)) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu), which in wild-type strains are modified with mcm(5)s(2)U, partially suppress phenotypes of an elp3 Delta strain. Objectives: To identify metabolic alterations in an elp3 Delta strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). Method: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3 Delta strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). The temperature sensitive elp3 Delta strain derivatives were cultivated at permissive (30 degrees C) or semi-permissive (34 degrees C) growth conditions. Results: Culturing an elp3 Delta strain at 30 or 34 degrees C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3D strain carrying the wild-type ELP3 gene. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu) suppressed a subset of the metabolic alterations observed in the elp3 Delta strain. Conclusion: Our results suggest that the presence of ncm(5)- and mcm(5)-side chains on wobble uridines in tRNA are important for metabolic homeostasis