Specific cleavage of pre-edited mRNAs in trypanosome mitochondrial extracts.

Abstract

RNA editing in Trypanosoma brucei is a posttranscriptional processing event that results in the addition and deletion of uridine residues within several mitochondrial mRNAs. We have examined reactions involving pre-edited precursor RNAs in vitro. In this study, we report specific cleavage of pre-edited cytochrome b (CYb), cytochrome oxidase subunit II (COII), and cytochrome oxidase subunit III (COIII) mRNAs when incubated with T. brucei mitochondrial extracts. The pre-edited CYb RNA was cleaved near the 3'-most uridine addition sites, within the region where editing would be expected to commence. Pre-edited COII mRNA was similarly cleaved adjacent to its small editing domain, while pre-edited COIII RNA was cleaved at multiple sites in the region where uridine addition and deletion occurs in vivo. In contrast, edited versions of CYb, COII, and COIII RNAs were not cleaved within the editing domains. Such differential cleavage of the edited and pre-edited forms of these mRNAs suggests either a direct involvement in RNA editing or involvement in another aspect of mitochondrial gene expression requiring cleavage of pre-edited RNAs.RNA editing in kinetoplastid mitochondria involves the addition and, to a lesser degree, deletion of uridine (U) residues at defined sites within several mRNAs (reviewed in references 3, 11, and 24). RNA editing is posttranscriptional, involving the modification of pre-edited precursor RNAs (16). Editing appears to be functionally important in the correction of a frameshift mutation in the cytochrome oxidase subunit II (COII) mRNA (4), in generation of initiation codons near the 5' ends of cytochrome b (CYb) and maxicircle unidentified reading frame 2 mRNAs (13,14), and in creation of nearly the entire coding regions of the cytochrome oxidase subunit III (COIII), ATPase subunit 6, and NADH dehydrogenase subunit 7 mRNAs (5, 12, 17). The variation in the degree to which different mRNAs are edited is extreme. For example, COII mRNA has only four U's added, while completely edited COIII mRNA contains 558 added U's and has 40 U's deleted (11).The information required for formation of edited mRNAs from pre-edited precursors is found in small (-70-nucleotide [nt]) guide RNAs (gRNAs) (6). gRNAs from maxicircle (6) and minicircle (21, 26) mitochondrial DNAs exist in the steady-state RNA population, but their precise role in editing is unknown.Based on the structure of gRNA and the presence of mitochondrial terminal uridylyl transferase (TUTase) and RNA ligase activities, Blum et al. (6) proposed the following mechanism for RNA editing. First, a gRNA hybridizes to mRNA sequences 3' to an editing site. An endoribonuclease activity then cleaves the pre-edited RNA once at the site where the complementarity between the pre-edited mRNA and gRNA ends. The TUTase and RNA ligase activities then insert the U residues that are directed by the gRNA and rejoin the RNA chain. Decker and Sollner-Webb (10) more random U additions within defined regions of the pre-edited RNA. Homology between correctly edited seque...

Purification and Characterization of MAR1

“…The puri- fied MAR1 has an absolute requirement for divalent cations and migrated in an SDS acrylamide gel as a 22-kDa band. This enzyme may correspond to previously described mitochondrial endoribonucleases from L. tarentolae and T. brucei (17,18). The L. tarentolae nuclease activity had an estimated molecular mass between 10 and 30 kDa, had an absolute divalent cation requirement, and showed a major cleavage site of pre-edited Cyb mRNA 2 nucleotides upstream of editing site 1 in addition to several other cleavages throughout the PER (18).…”

“…However, the L. tarentolae activity also showed a stimulation by heparin or by digestion of the crude lysate with proteinase K and an inhibition by adenylate nucleotides or GTP. The T. brucei nuclease activity showed a relatively high thermal stability, had a specificity for sites within the PER's of Cyb, cytochrome oxidase II, and cytochrome oxidase III mRNA substrates, and did not cleave within the fully edited regions of the same RNAs (17). Both activities were only characterized from crude mitochondrial lysates.…”

“…One of these activities, which sedimented at 20 S in glycerol gradients, exhibited a gRNA-dependent cleavage at the first mismatch upstream of a duplex RNA region (14 -16), precisely as predicted by the enzyme cascade model for RNA editing (12). Another activity, which sedimented at 15 S and was independent of added gRNA for cleavage, might correspond to an endoribonuclease activity that has been described previously in crude mitochondrial extracts from both L. tarentolae and T. brucei (17,18). The endoribonuclease activities in the crude extracts were both shown to cleave pre-edited Cyb mRNAs two nucleotides upstream of the first editing site.…”

RNA editing in kinetoplastid protozoa

“…This model requires gRNA-independent endonucleolytic cleavage of the pre-mRNA. An activity has been identified in trypanosome mt which could perform this role (41,67). However, it has not been established whether this activity plays a role in editing.…”

RNA editing in trypanosomes

“…There are also some arguments in favour of an enzymic pathway. First, an RNA ligase and (an) endonuclease(s) which cleave RNAs in the vicinity of editing sites have indeed been found in (mitochondria of) L. tarentolae and T brucei [63,64,87,881. These enzymes played a central role in older 'enzyme cascade' models of RNA editing [52] that were in vogue before the existence of chimeric molecules had been demonstrated.…”