A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis.
We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3'-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3'-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes.
Morillo-Huesca
Macarena
M
Departamento de Genética, Universidad de Sevilla, Seville, Spain.
Vanti
Manuela
M
Chávez
Sebastián
S
D000135:Acid Phosphatase; D001681:Biological Assay; D009154:Mutation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; D000135:Acid Phosphatase; C488916:PHO5 protein, S cerevisiae