Dominant genetic screen for cofactors that enhance antisense RNA-mediated gene silencing in fission yeast.
Specific gene silencing has been demonstrated in a number of organisms by the introduction of antisense RNA. Mutagenesis of host-encoded factors has begun to unravel the mechanism of several forms of RNA-mediated gene silencing and has suggested that it may have been conserved through evolution. This has led to the identification of certain host genes, which, when mutated, abrogate this phenomenon. Conversely, the identification of other factors that, when co-expressed or overexpressed, can enhance gene inhibition is equally important for both elucidating the mechanism of this process and enhancing gene silencing in recalcitrant systems. We have taken such a dominant genetic approach to identify several host-encoded factors that dramatically enhance target gene silencing when co-expressed with antisense RNA in fission yeast. The transcription factor thi1 and, surprisingly, the ATP-dependent RNA helicase ded1 were initially shown to enhance gene silencing in this system. Additionally, screening of a Schizosaccharomyces pombe cDNA library identified four novel antisense-enhancing sequences (aes factors) all of which are homologous to genes encoding proteins with natural affinities for nucleic acids. These findings demonstrate the utility of this strategy in identifying host-encoded factors that can modulate gene silencing when co-expressed with antisense RNA and possibly other forms of gene-silencing activators.
Raponi
Mitch
M
Department of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW 2052, Australia.
Arndt
Greg M
GM
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D053487:DEAD-box RNA Helicases; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D020868:Gene Silencing; D005800:Genes, Fungal / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D020365:RNA Helicases; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D016372:RNA, Antisense; D012331:RNA, Fungal; D012333:RNA, Messenger; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C086956:thi1 protein, S pombe; C492151:sum3 protein, S pombe; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases