Isolation and Characterization of a U-specific 3′-5′-Exonuclease from Mitochondria of Leishmania tarentolae

Ruslan Aphasizhev,Larry Simpson

doi:10.1074/jbc.m100297200

Abstract

We have purified a 3'-5'-exoribonuclease from mitochondrial extract of Leishmania tarentolae over 4000-fold through six column fractionations. This enzyme digested RNA in a distributive manner, showed a high level of specificity for 3'-terminal Us, and was blocked by a terminal dU; there was slight exonucleolytic activity on a 3'-terminal A or C but no activity on a 3'-terminal G residue. The enzyme preferred single-stranded 3'-oligo(U) overhangs and did not digest duplex RNA. Two other 3'-5'-exoribonuclease activities were also detected in the mitochondrial extract, one of which was stimulated by a 3'-phosphate and the other of which degraded RNAs with a 3'-OH to mononucleotides in a processive manner. The properties of the distributive U-specific 3'-5'-exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.

Highlights

3Ј-5Ј-Exoribonucleases have been shown to play important roles in the maturation and degradation of RNAs, including the 3Ј-end maturation of the 5.8 S rRNA [1] and pre-tRNAs [2] and both deadenylation-dependent [3] and deadenylation-independent mRNA decay in eukaryotes [4]
The exonuclease activity was proposed to be a reversal of a 3Ј-terminal uridylyltransferase (TUTase)1 that adds Us to the 3Ј-end of the 5Ј-cleavage fragment at a U-insertion editing site [19], but this was disproved by showing that pyrophosphate inhibits TUTase but does not inhibit the exonuclease activity [16]
In this paper we report the partial purification and characterization of a U-specific 3Ј-5Ј-exonuclease activity from L. tarentolae mitochondria, which represents a candidate for the enzyme involved in removal of the Us in U-deletion RNA editing

Summary

Introduction

3Ј-5Ј-Exoribonucleases have been shown to play important roles in the maturation and degradation of RNAs, including the 3Ј-end maturation of the 5.8 S rRNA [1] and pre-tRNAs [2] and both deadenylation-dependent [3] and deadenylation-independent mRNA decay in eukaryotes [4]. The properties of the distributive U-specific 3؅-5؅-exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.

Results

Conclusion