The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria

Abstract

Recent advances in in vitro systems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3′-end of gRNA to the editing site appear to be untenable. Two types of in vitro systems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3′-end of the 5′-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3′-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitro evidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.

On the Possibility of Constructive Neutral Evolution

“…In trypanosome mitochondria, gene expression proceeds through an eleborate stage in which insertion of U (uracil) nucleotides (nt) occurs at thousands of specific sites in protein-coding regions of gene transcripts (Alfonzo et al 1997). This process does not destroy the meaning of the genetic message but, instead, creates it: the unedited transcripts are encoded in bizarre "cryptogenes" (e.g., see Fig.…”

“…2A). Once a gRNA is anchored to its template by base-pairing, editing of a short block of transcript (usually 30-40 nt) proceeds by cycles of cleavage, U-insertion, and ligation (Alfonzo et al 1997).…”

“…The editing machinery exhibits four biochemical activities found commonly in other cells: RNA helicase (to wind or unwind duplexes), endoribonuclease (to cleave an RNA strand), terminal uridyl transferases (to add uridine residues to a strand), and RNA ligase (to ligate strands) (Alfonzo et al 1997). Prior to participating in RNA editing, some or all of these activities may have operated together in other contexts.…”

Sequence bias in edited kinetoplastid RNAs

“…The single-celled flagellated organisms in the order Kinetoplastidae posttranscriptionally remodel the sequences of many of their mitochondrially encoded transcripts by the precise insertion and deletion of uridylates (Us)+ These sequence changes are directed by small trans-acting guide RNAs (gRNAs) that are complementary (by both Watson-Crick and G:U base pairing) to edited sequences (Blum et al+, 1990; for recent reviews, see Alfonzo et al+, 1997;Kable et al+, 1997;Sloof & Benne, 1997)+ The insertion and deletion of Us in kinetoplastid RNAs (kRNAs) creates the functional open reading frames as is evident from the homology of the proteins predicted from translation of edited mRNAs to proteins encoded in the mitochondria of numerous other organisms+ In the kinetoplastid parasite, Trypanosoma brucei, 12 of the 18 mitochondrially encoded pre-mRNAs are edited to varying degrees, many extensively, resulting in a total of 1,362 editing sites (Arts & Benne, 1996, and references therein)+ This quantity of editing is predicted to require hundreds of gRNAs, each specifying the editing of 4 to 10 sites and resulting in complementarity to an ;45-nt stretch of edited RNA+ Although only a small fraction of gRNAs have been sequenced, renaturation kinetic analyses of the coding capacity of the minicircle component of mitochondrial DNA of T. brucei predicts more than enough capacity for the number of different gRNAs required to specify all the observed editing (Stuart, 1993)+ The mechanism by which gRNAs direct the insertion and deletion of Us follows a cleavage/U addition or removal/ligation pathway initially proposed by Blum et al+ (1990)+ gRNAs appear to select the site to be processed by forming a short duplex adjacent to the editing site+ Editing is initiated by endonucleolytic cleavage of the pre-mRNA 39 (with respect to gRNA) to the terminus of this duplex, generating upstream and downstream pre-mRNA cleavage fragments+ For clarity, the positions that are 59 and 39, with respect to pre-mRNA, of the editing site will be referred to as upstream and downstream, respectively+ After cleavage, the 39 terminus of the upstream fragment is processed by U addition or removal during insertion and deletion editing, respectively, and then the fragments are rejoined+ Each round of editing at a site in vitro is specified by the gRNA sequence opposing it (Cruz-Reyes & SollnerWebb, 1996;Kable et al+, 1996;Seiwert et al+, 1996)+ During U deletion, the cleavage occurs downstream of the Us to be deleted (Cruz-Reyes & Sollner-Webb, 1996;Siewert et al+, 1996), and the Us are removed by a U-specific 39 exonuclease until the first non-U nucleotide is encountered (S+ Lawson, unpubl+ data)+ Thus, the site of gRNA-directed cleavage and the specificity of the exonuclease can account for the number of Us deleted+ How the gRNA directs the addition of the appropriate number of Us during insertion editing appears more complex+ Insertion sites are edited by insertion of anywhere from 1 to 13 Us (Benne, 1994)+ Accuracy in the number of Us inserted is e...…”

The Effect of RNA Interference Down-regulation of RNA Editing 3′-Terminal Uridylyl Transferase (TUTase) 1 on Mitochondrial de Novo Protein Synthesis and Stability of Respiratory Complexes in Trypanosoma brucei

“…Gene expression in the kinetoplast-mitochondrion of trypanosomatid protists involves a unique post-transcriptional mRNA maturation process, termed RNA editing, whereby Uresidues are inserted to or deleted from a pre-edited transcript, and a translatable reading frame is thus created (1)(2)(3)(4)(5). 12 of the 18 protein-coding genes in the maxicircle component of the mitochondrial or kinetoplast DNA in Trypanosoma brucei or Leishmania tarentolae encode transcripts that require varying degrees of editing for translation competence (6).…”