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J. Biol. Chem., Vol. 276, Issue 20, 16919-16930, May 18, 2001
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From the Department of Immunology, the University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030
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.
Localization and Stability of Introns Spliced from the
Pem Homeobox Gene*
*
This work was supported by National Institutes of Health
Grant CA78023 and National Science Foundation Grant MCB-9307963.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Immunology,
the University of Texas M. D. Anderson Cancer Center, Box 180, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-794-5526; Fax:
713-745-0846; E-mail: mwilkins@mail.mdanderson.org.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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