RNA splicing and debranching viewed through analysis of RNA lariats.

摘要: Intron lariat RNAs, created by pre-mRNA splicing, are sources of information on gene expression and structure. Although produced equivalently to their corresponding mRNAs, the vast majority intron RNAs rapidly degraded. However, levels enhanced in cells deficient for RNA debranching enzyme, which catalyzes linearization these rate-limiting step degradation. Furthermore, lariats resistant degradation 3′ exonuclease polynucleotide phosphorylase (PNPase), providing a means enrich RNAs. Working with yeast Saccharomyces cerevisiae as model organism, our goal was develop novel combinations methods enhance use objects study. Using RT-PCR assays developed detecting quantifying specific we demonstrate resistance PNPase sensitivity cleavage enzyme. We also employ sequential treatments two enzymes produce characteristic effects linear establish utility analyzing enzyme variants vitro reactions discuss several possible applications, including measuring relative rates transcription combining non-gene-specific sequencing approach genome annotation. In summary, enzymatic that combined or sequencing, can be powerful tools advance studies expression, alternative any process depends