In vitro uridine insertion RNA editing mediated by cis-acting guide RNAs

Uridine (U) insertion/deletion editing of mitochondrial mRNAs in kinetoplas tid protozoa is a posttranscriptional process mediated by guide RNAs (gRNAs ), The gRNAs direct the precise insertion and deletion of Us by a cleavage- ligation mechanism involving base pairing, We show that a cognate gRNA in c is at the 3' end of a preedited NADH dehydrogenase 7 (ND7) mRNA substrate c an direct U insertions at editing site 1 when incubated with a mitochondria l lysate from Leishmania tarentolae, The efficiency of gRNA-dependent U ins ertion mediated by a cis-acting gRNA is greater on a molar basis than that for a trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interacti on, Blocking the 3' end of a cis-acting gRNA lacking a 3' oligo[U] tail has no effect on gRNA-dependent U insertions, nor does providing the gRNA in c is upstream of the mRNA, confirming the previous observation that the termi nal 2'- and 3'-hydroxyls of the gRNA are not involved in U insertion activi ty, These results also establish that the oligo[U] tail is not required for U insertion in vitro. Increasing the extent of base pairing between the 3' end of the gRNA and the 5' end of the mRNA significantly increases in vitr o gRNA-dependent U insertion at site 1, presumably by maintaining the mRNA 5' cleavage fragment within the editing complex, We speculate that, in vivo , protein:RNA and/or protein:protein interactions may be responsible for ma intaining the mRNA 5' cleavage fragment in close proximity to the mRNA 3' c leavage fragment, and that such interactions may be rate limiting in vitro.