Uridylate-specific 3' 5'-exoribonucleases involved in uridylate-deletion RNA editing in trypanosomatid mitochondria.
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.
Rogers
Kestrel
K
Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Gao
Guanghan
G
Simpson
Larry
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015536:Down-Regulation; D005095:Exoribonucleases / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D018320:Leishmania major / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D017393:RNA Editing; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000378:metabolism*
D011994:Recombinant Proteins; D014542:Uridine Monophosphate; D005095:Exoribonucleases