Thiolation controls cytoplasmic tRNA stability and acts as a negative determinant for tRNA editing in mitochondria.
Department of Microbiology and The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
Abstract
Kinetoplastids
encode a single nuclear tryptophanyl tRNA that contains a CCA anticodon
able to decode the UGG codons used in cytoplasmic protein synthesis but
cannot decode the mitochondrial UGA codons. Following mitochondrial
import, this problem is circumvented in Trypanosoma brucei by
specifically editing the tRNA(Trp) anticodon to UCA, which can now
decode the predominant mitochondrial UGA tryptophan codons. This tRNA
also undergoes an unusual thiolation at position 33 of the anticodon
loop, the only known modification at U33 in any tRNA. In other
organisms, tRNA thiolation is mediated by the cysteine desulfurase, Nfs1
(IscS). However, T. brucei encodes two Nfs homologues, one cytoplasmic
and the other mitochondrial. We show by a combination of RNA
interference and Northern and Western analyses that the
mitochondria-targeted TbNfs, and not TbNfs-like protein, is essential
for thiolation of both cytosolic and mitochondrial tRNAs. Given the
exclusive mitochondrial localization of TbNfs, how it mediates
thiolation in the cytoplasm remains unclear. Furthermore, thiolation
specifically affects thiolated tRNA stability in the cytoplasm but more
surprisingly acts as a negative determinant for the essential C to U
editing in T. brucei. This provides a first line of evidence for
mitochondrial C to U editing regulation in this system.
