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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
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Polyadenylation Regulates the Stability of Trypanosoma brucei Mitochondrial RNAs*

Christopher M. Ryan {ddagger}, Kevin T. Militello {ddagger} § 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, 731–742). 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.

{ddagger} 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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