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Design and Application of Temperature Sensitive Mutants in Essential Factors of RNA Splicing and RNA Interference Pathway in Schizosaccharomyces Pombe

Electronic Theses of Indian Institute of Science

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Title Design and Application of Temperature Sensitive Mutants in Essential Factors of RNA Splicing and RNA Interference Pathway in Schizosaccharomyces Pombe
 
Creator Nagampalli, Vijay Krishna
 
Subject RNA Splicing
Cell Cycle
Schizosaccharomyces Pombe
RNA Interference
RNA Transcription
Temperature Sensitive Mutants
Fission Yeast Genes
Missense Mutants
Fission Yeast Pre-mRNA Splicing
Heterochromatin
Yeast SpPrp18
Cell Cycle Progression
Yeast SpSlu7
Microbiology and Cell Biology
 
Description Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast.
Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18
Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns.
In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures
 
Contributor Vijayraghavan, Usha
Varadarajan, Raghavan
 
Date 2018-05-10T05:44:50Z
2018-05-10T05:44:50Z
2018-05-10
2014
 
Type Thesis
 
Identifier http://etd.iisc.ernet.in/2005/3515
http://etd.iisc.ernet.in/abstracts/4382/G26707-Abs.pdf
 
Language en_US
 
Relation G26707