Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae

Abstract

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5 of the editing sites and guanosines immediately 3 of guiding nucleotides. U insertions are directed both 5 and 3 of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

The 3′-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA

“…Several in vitro studies have shown that when transacting gRNAs are presented in cis they faithfully direct the addition or deletion of uridines at specific sites within the mRNA (Kapushoc and Simpson 1999;Cruz-Reyes et al 2001). Additionally, the 5Ј UTR of some pre-edited mRNAs are known to enhance the activity of nonspecific uridine addition and deletion in adjacent sequences within the mRNA (Connell et al 1997;Pai et al 2003). While highlighting the ability of editing events to be influenced by cis regions, these studies have not directly addressed the ability of a 3Ј UTR to function as a cis-acting guide.…”

An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction

“…The 39 end of the RNA substrate had been blocked prior to the assay through ligation to the 59 end by treatment with RNA ligase. This circular substrate RNA ensured that the higher-mobility product did not result from the addition of U's to the 39 end by a terminal uridyyl transferase (TUTase) activity that is present within the editing extract (Brown et al 1999;Pai et al 2003).…”

“…This is not an intrinsic deficiency of the editing extract as other RNAs are accurately edited by the same in vitro reaction . Rather, it is most likely a reflection of the sequence that was selected upstream of the editing site, which was previously shown to significantly influence editing fidelity (Igo et al 2002;Pai et al 2003). There was not an explicit requirement during the selection to have completely accurate editing provided that any U insertions still triggered a conformational change that activated streptavidin binding.…”

“…The disruption of the helix by the selected deletions possibly makes the editing site more accessible to the editing complex. The sequence and structure upstream of the editing site have previously been shown to influence editing efficiency (Igo et al 2002;Pai et al 2003), and as a result it is not surprising that the mutations of U-31 and A-32 also have major effects on editing. All of the other mutations within the streptavidin-binding aptamer had some type of minor effect on editing efficiency, suggesting that alterations in overall tertiary structure could influence the reaction (Table 2).…”

“…High-throughput screening of chemical libraries is an alternative strategy to identify novel drugs inhibiting the editing reaction, but it also has limitations. These are primarily related to the in vitro editing assays not having the sensitivity and format necessary for high-throughput screening to be practical and economically feasible (Byrne et al 1996;Kable et al 1996;Seiwert et al 1996;Wang et al 2002;Pai et al 2003). A novel assay is described here that can detect edited product in the low femtomole range and is ideal for high-throughput format.…”