RESOLUTION OF THE RNA EDITING GRNA-DIRECTED ENDONUCLEASE FROM 2 OTHERENDONUCLEASES OF TRYPANOSOMA-BRUCEI MITOCHONDRIA

RNA editing in kinetoplastids, the specific insertion and deletion of U residues, requires endonuclease cleavage of the pre-mRNA at each cyc le of insertion/deletion. We have resolved three endoribonuclease acti vities from Trypanosoma brucei mitochondrial extracts that cleave CYb pre-mRNA specifically. One of these, which sediments at similar to 20S and is not affected substantially by DTT, has all the features of the editing endonuclease. It cleaves CYb pre-edited or partially edited m RNA only when annealed to the anchor region of a cognate guide RNA (SR NA), and it cleaves accurately just 5' of the duplex region. Its speci ficity is for the 5' end of extended duplex RNA regions, and this prev ents cleavage of the SRNA or other positions in the mRNA. This gRNA-di rected nuclease is evidently the same activity that functions in A6 pr e-mRNA editing. However, it is distinct and separable from a previousl y observed DTT-requiring endonuclease that sediments similarly under c ertain conditions, but does not cleave precisely at the first editing site in either the presence or absence of a gRNA. The editing nuclease is also distinct from a DTT-inhibited endonuclease that cleaves numer ous free pre-mRNAs at a common structure in the region of the first ed iting site.