Localization and stability of introns spliced from the Pem homeobox gene

RNA splicing generates two products in equal molar amounts, mature mRNAs an d spliced introns, Although the mechanism of RNA splicing and the fate of t he spliced mRNA products have been well studied, very little is known about the fate and stability of most spliced introns, Research in this area has been hindered by the widely held view that most vertebrate introns are too unstable to be detectable. Here, we report that we are able to detect all t hree spliced introns from the coding region of the Pem homeobox gene. By us ing a tetracycline (tet)-regulated promoter, we found that the half-lives o f these Pem introns ranged from 9 to 29 min, comparable with those of short lived mRNAs such as those encoding c-fos and c-myc, The half-lives of the Pem introns correlated with both their length and 5' to 3' orientation in t he Pem gene. Subcellular fractionation analysis revealed that spliced Pem i ntrons and pre-mRNA accumulated in the nuclear matrix, high salt-soluble, a nd DNase-sensitive fractions within the nucleus. Surprisingly, we found tha t all three of the spiced Pem introns were also in the cytoplasmic: fractio n, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from a nother gene were virtually excluded from this fraction. This indicates eith er that spliced Pem introns are uniquely exported to the cytoplasm for degr adation or they reside in a unique soluble nuclear fraction. Our study has implications for understanding the regulation of RNA metabolism, as the sta bility of introns and the location of their degradation may dictate the fol lowing: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are a vailable for further rounds of transcription, and (iii) the rate at which s plicing factors are recycled.