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Originally published In Press as doi:10.1074/jbc.M303552200 on June 11, 2003
J. Biol. Chem., Vol. 278, Issue 35, 32753-32762, August 29, 2003
Polyadenylation Regulates the Stability of Trypanosoma brucei Mitochondrial RNAs*
Christopher M. Ryan ,
Kevin T. Militello and
Laurie K. Read ¶
From the
Department of Microbiology and the Witebsky Center for Microbial
Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, State
University of New York, Buffalo, New York 14214
Polyadenylation of RNAs plays a critical role in modulating rates of RNA
turnover and ultimately in controlling gene expression in all systems examined
to date. In mitochondria, the precise mechanisms by which RNAs are degraded,
including the role of polyadenylation, are not well understood. Our previous
in organello pulse-chase experiments suggest that poly(A) tails
stimulate degradation of mRNAs in the mitochondria of the protozoan parasite
Trypanosoma brucei (Militello, K. T., and Read, L. K. (2000) Mol.
Cell. Biol. 21, 731742). In this report, we developed an in
vitro assay to directly examine the effects of specific
3'-sequences on RNA degradation. We found that a salt-extracted
mitochondrial membrane fraction preferentially degraded polyadenylated
mitochondrially and non-mitochondrially encoded RNAs over their non-adenylated
counterparts. A poly(A) tail as short as 5 nucleotides was sufficient to
stimulate rapid degradation, although an in vivo tail length of 20
adenosines supported the most rapid decay. A poly(U) extension did not promote
rapid RNA degradation, and RNA turnover was slowed by the addition of uridine
residues to the poly(A) tail. To stimulate degradation, the poly(A) element
must be located at the 3' terminus of the RNA. Finally, we demonstrate
that degradation of polyadenylated RNAs occurs in the 3' to 5'
direction through the action of a hydrolytic exonuclease. These experiments
demonstrate that the poly(A) tail can act as a cis-acting element to
facilitate degradation of T. brucei mitochondrial mRNAs.
Received for publication, April 5, 2003
, and in revised form, May 19, 2003.
* This work was supported in part by National Institutes of Health Grant
AI47329 (to L. K. R.). The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Supported in part by National Institutes of Health Training Grant
AI07614.
Present address: Dept. of Immunology and Infectious Disease, Harvard School
of Public Health, Boston, MA 02115.
¶
To whom correspondence should be addressed: Dept. of Microbiology, School of
Medicine and Biomedical Sciences, SUNY, 138 Farber Hall, 3435 Main St.,
Buffalo, NY 14214. Tel.: 716-829-3307; Fax: 716-829-2158; E-mail:
lread{at}acsu.buffalo.edu.

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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
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