The stability and fate of a spliced intron from vertebrate cells

Introns constitute most of the length of typical pre-mRNAs in vertebrate ce lls. Thus, the turnover rate of introns may significantly influence the ava ilability of ribonucleotides and splicing factors for further rounds of tra nscription and RNA splicing, respectively. Given the importance of intron t urnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1(C beta 1) the first intron from the constant region of the mouse T- cell receptor-beta (TCR-beta) gene. Using a tetracycline (tet)-regulated pr omoter, we demonstrate that spliced IVS1(C beta 1) and its pre-mRNA had hal f-lives of 6.0 +/- 1.4 min and 3.7 +/- 1.0 min, respectively. We also exami ned the half-lives of these transcripts by using actinomycin D (Act.D). Act .D significantly stabilized IVS1(C beta 1) and its pre-mRNA, suggesting tha t Act.D not only blocks transcription but exerts rapid and direct posttrans criptional effects in the nucleus. We observed that in vivo spliced IVS1(C beta 1) accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1(C beta 1) as a lariat did not result from an intrinsic inability to be debranched, as it could be deb ranched in vitro, albeit somewhat less efficiently than an adenovirus intro n. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1(C beta 1) lariats cofractionated with pre-mRNA, but not alway s with mRNA in the nucleus. Some IVS1(C beta 1) also appeared to be selecti vely exported to the cytoplasm, whereas TCR-beta pre-mRNA remained in the n ucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.