Yeast Pre-mRNA Is Composed of Two Populations with Distinct Kinetic Properties

Abstract

As an approach to the study of yeast pre-mRNA splicingin vivo,we have examined properties of transcripts derived from a gal-UAS intron-containing fusion gene encoding RP51A and a series of its derivatives. RNA half-life measurements were carried out after transcription initiation was blocked by the addition of glucose. Pre-mRNA encoded by GalRP51A decayed with a half-life of ∼6 min and was substantially polyadenylated, and transcripts derived from a nonspliced version of the same gene decayed with a similar half-life (∼4 min). A comparison of the steady-state levels of these two transcripts suggests that the bulk of GalRP51A pre-mRNA is processed much more rapidly, with an average lifetime of about 2 s. We propose that this inferred population of rapidly processed molecules is spliced cotranscriptionally and that it is the principal precursor to GalRP51A mRNA. Although the pre-mRNA molecules detected are therefore unlikely to be the major splicing precursors, anin vivoassay suggests that they are likely to have bound splicing factors. They must then be spliced much more slowly than most primary transcripts, or not spliced at all and then degraded through a different cellular pathway. As a result of its comparatively long lifetime, this minor fraction of the pre-mRNA population makes up the majority of the steady-state level of GalRP51A pre-mRNA.