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Abstract
RNA splicing generates two products in equal molar amounts, mature mRNAs and spliced introns. Although the mechanism of RNA splicing and the fate of the 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 three spliced introns from the coding region of the Pem homeobox gene. By using a tetracycline (tet)-regulated promoter, we found that the half-lives of 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 the Pem gene. Subcellular fractionation analysis revealed that spliced Pem introns and pre-mRNA accumulated in the nuclear matrix, high salt-soluble, and DNase-sensitive fractions within the nucleus. Surprisingly, we found that all three of the spliced Pem introns were also in the cytoplasmic fraction, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from another gene were virtually excluded from this fraction. This indicates either that spliced Pem introns are uniquely exported to the cytoplasm for degradation or they reside in a unique soluble nuclear fraction. Our study has implications for understanding the regulation of RNA metabolism, as the stability of introns and the location of their degradation may dictate the following: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are available for further rounds of transcription, and (iii) the rate at which splicing factors are recycled.
Highlights
Stead, evidence suggests that most introns in modern organisms arose serendipitously as a result of the following two processes: the shuffling of small intronless primordial genes to generate large genes with introns, and the introduction of introns into intronless genes by transposition-type events [7]
Our study has implications for understanding the regulation of RNA metabolism, as the stability of introns and the location of their degradation may dictate the following: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are available for further rounds of transcription, and (iii) the rate at which splicing factors are recycled
We found that U6 small nuclear RNA (snRNA) was primarily restricted to the nuclear fraction; little or none was detected in the nuclear wash or cytoplasmic fractions of all six rat cell lines (Fig. 5C and data not shown; note that a weak cytoplasmic U6 snRNA signal was seen in 208F cells, this lane was overloaded)
Summary
The mechanism of RNA splicing and the fate of the 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. We found that all three of the spliced Pem introns were in the cytoplasmic fraction, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from another gene were virtually excluded from this fraction. Given the probable importance of intron turnover, it is surprising that very little is known about this topic In part, this deficiency may reflect the fact that very few spliced nuclear pre-mRNA introns have been detected in vertebrate cells. These findings may have important implications for RNA metabolism in general
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