Biomedical subjects

Gregory J Connell

Publications and source records attributed to Gregory J Connell.

4 recordsLinked to original sources

PubMed2003

Cis-acting elements stimulating kinetoplastid guide RNA-directed editing.

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.

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PubMed2003

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

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.

Animals↗

PubMed2002

Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract.

The coding sequence within several mitochondrial mRNAs of the trypanosomatid protozoa is created through editing by the precise insertion and deletion of U nucleotides. The biochemical characterisation of the editing reaction in the Leishmania genus of the trypanosomatids has been hindered by the lack of a direct in vitro assay. We describe here the first direct assay for the detection of guide RNA-directed editing mediated by a mitochondrial extract prepared from two independent isolates of Leishmania tarentolae. The assay enabled the editing activity within a L. tarentolae mitochondrial extract to be significantly enriched and will facilitate the characterisation of the editing reaction. The results suggest that the difficulty in establishing an assay for the L. tarentolae reaction was not simply a result of the catalytic machinery being limiting but rather reflected the presence of constraints on both the guide RNA and mRNA sequences.

Animals↗

PubMed2002

pH dependent conformational changes within the iron responsive element.

The expression of several mRNAs related to iron importation, storage and utilization within mammalian cells are regulated through interactions of iron regulatory proteins with an iron responsive element, an RNA hairpin with a bulged C. A dimethylsulfate modification interference assay was used to demonstrate that the iron responsive element undergoes significant pH dependent conformational changes. Specifically, it was demonstrated that the phylogenetically conserved A within the hairpin loop and an intra-loop C-G base pair are highly sensitive to changes in pH. The conserved C of the bulged loop does not significantly affect the pH dependent conformational changes of the hairpin loop. These studies have structural implications for an RNA-protein interaction that is critical to mammalian iron regulation.

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