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J. Biol. Chem., Vol. 282, Issue 40, 29073-29080, October 5, 2007

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Uridylate-specific 3'–5'-Exoribonucleases Involved in Uridylate-deletion RNA Editing in Trypanosomatid Mitochondria^*

From the Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095

In kinetoplastid protists, maturation of mitochondrial pre-mRNAs involves the insertion and deletion of uridylates (Us) within coding regions, as specified by mitochondrial DNA-encoded guide RNAs. U-deletion editing involves endonucleolytic cleavage of the pre-mRNA at the editing site followed by U-specific 3'–5'-exonucleolytic removal of nonbase-paired Us prior to ligation of the two mRNA cleavage fragments. We showed previously that an exonuclease/endonuclease/phosphatase (EEP) motif protein from Leishmania major, designated RNA editing exonuclease 1 (REX1) (Kang, X., Rogers, K., Gao, G., Falick, A. M., Zhou, S.-L., and Simpson, L. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1017–1022), exhibits 3'–5'-exonuclease activity. Two EEP motif proteins have also been identified in the Trypanosoma brucei editing complex. TbREX1 is a homologue of LmREX1, and TbREX2 shows homology to another editing protein in L. major, which lacks the EEP motif (LmREX2*). Here we have expressed the T. brucei EEP motif proteins in insect cells and purified them to homogeneity. We showed that these are U-specific 3'–5'-exonucleases that are inhibited by base pairing of 3' Us. The recombinant EEP motif alone also showed 3'–5' U-specific exonuclease activity, and mutations of the REX EEP motifs greatly reduced exonuclease activity. The absence of enzymatic activity in LmREX2* was confirmed with a purified recombinant protein. We showed that pre-cleaved U-deletion editing could be reconstituted with either TbREX1 or TbREX2 in combination with either RNA ligase, LmREL1, or LmREL2. Down-regulation of TbREX2 expression by conditional RNA interference had little effect on parasite viability or sedimentation of the L-complex, suggesting either that TbREX2 is inactive in vivo or that TbREX1 can compensate for the loss of TbREX2 function in down-regulated cells.

Received for publication, June 4, 2007 , and in revised form, August 13, 2007.

^* This work was supported by National Institutes of Health Grant AI09102 (to L. S.) and by Ruth L. Kirschstein National Research Service Award GM07185 (to 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.

¹ Present address: Kurland Heart Institute, 200 Med Plaza, Los Angeles, CA 90095.

² To whom correspondence should be addressed: David Geffen School of Medicine at UCLA, 1602 Molecular Science Bldg., Los Angeles, CA 90095-1489. Fax: 310-341-2271; E-mail: simpson{at}kdna.ucla.edu.

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