Two distinct roles for terminal uridylyl transferases in RNA editing.

Content Provider	Semantic Scholar
Author	Gott, Jonatha
Copyright Year	2003
Abstract	G ene expression in the mitochondria of kinetoplastid protists like the human pathogen Trypanosoma brucei occurs via a remarkable pathway involving both the deletion of encoded uridines (Us) and the insertion of extra Us at defined positions within mRNAs synthesized from ‘‘cryptogenes’’ in the maxicircle DNA (see refs. 1 and 2 for recent reviews). In some cases, 50% of the nucleotides present in the mature mRNA are generated by RNA editing events. U addition and removal create sense from nonsense, producing ORFs that encode the proteins involved in oxidative phosphorylation and other mitochondrial functions. Many of these changes are developmentally regulated, occurring only in the procyclic (insect) stage or the bloodstream (mammalian) form. Although the overall mechanism by which this extraordinary phenomenon occurs has been sketched out (3–6), filling in the details has been difficult because of the low abundance and complexity of the editing apparatus, the low efficiency of in vitro editing assays, the lack of assays for processive editing, and difficulties in assigning functions to specific proteins. The article by Aphasizhev et al. (7) in this issue of PNAS provides important information regarding the function of two of the key enzymes involved in this fascinating process. The insertion and deletion of Us into kinetoplast mRNAs occur through the concerted action of a series of enzymes (Fig. 1) (see references in ref. 2). Specificity is provided by small RNA molecules termed guide RNAs (gRNAs), which base-pair to preedited mRNAs just downstream of editing sites (8). gRNAs have three functional domains: an anchor region, which anneals to the substrate; a guiding region, which directs the insertion or deletion of U residues; and an oligo(U) tail, which is added posttranscriptionally and is thought to help tether the purine-rich 5 cleavage fragment to the rest of the complex. Binding of the gRNA targets cleavage of the mRNA immediately upstream of the anchor duplex by an editing endonuclease, creating a 5 cleavage fragment that is the substrate for either a 3 terminal uridylyl transferase (TUTase) in the case of U insertion or a 3 to 5 exonuclease for U deletions. The number of Us added or deleted is determined by both the sequence of the gRNA and the specificity of the editing enzymes; the new 3 end of the upstream fragment pairs with the guiding region, extending the anchor duplex and directing ligation of the two mRNA fragments. Editing of a given message occurs with an overall 3 to 5 polarity and usually involves multiple gRNAs that act sequentially. Often gRNA binding sites are created by prior editing events and are presumably accessed upon dissociation of the previous gRNA. Editing is catalyzed by a multisubunit complex, termed the ‘‘editosome.’’ The size of this macromolecular complex varies, depending on the isolation conditions, but it generally sediments at 19– 25S in glycerol gradients and contains between 7 and 21 polypeptides (9–13), only some of which have been directly linked to the editing reaction (see refs. 1 and 2). A number of editosome components have now been identified through sequencing and or mass spectrometric analysis of proteins present in native complexes, and their genes have been cloned based on sequences present in the Leishmania major and T. brucei genome databases or via PCR using degenerate primers (13–16). This information has made it possible to augment conventional biochemical fractionation through purification of complexes containing individual tandem affinity purification-tagged proteins (15, 16), and the availability of antibodies raised against native complexes or recombinant proteins has facilitated affinity purification, immunodepletion, and coimmunopre-
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Page Count	4
File Format	PDF HTM / HTML
Alternate Webpage(s)	http://dna.kdna.ucla.edu/simpsonlab/Lab%20publications/tail2.pdf
Alternate Webpage(s)	http://www.pnas.org/content/100/19/10583.full.pdf
PubMed reference number	12963809v1
Volume Number	100
Issue Number	19
Journal	Proceedings of the National Academy of Sciences of the United States of America
Language	English
Access Restriction	Open
Subject Keyword	Binding Sites Biopolymer Sequencing Blood Circulation Chromatography, Affinity Clinical act of insertion Clone Cells Databases Deletion Mutation Encode (action) Exonuclease Genus: Leishmania Glycerin Insertion Mutation Leishmania major Ligation Mammals Nonsense mutation Nucleotides Open Reading Frames Oxidative Phosphorylation Pathogenic organism Polypeptides Purines RNA Editing RNA, Guide Recombinant Proteins Recombinants Review [Publication Type] Tracer Transferase endonuclease kinetoplast mRNA editing complex negative regulation of production of siRNA involved in RNA interference polarity protists
Content Type	Text
Resource Type	Article