Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22?kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA^Tyr is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.     

RNA editing sites in plant mitochondria can share cis-elements

RNA editing in flowering plant mitochondria alters numerous C nucleotides in a given mRNA molecule to U residues. To investigate whether neighbouring editing sites can influence each other we analyzed in vitro RNA editing of two sites spaced 30 nt apart. Deletion and competition experiments show that these two sites carry independent essential specificity determinants in the respective upstream 20-30 nucleotides. However, deletion of a an upstream sequence region promoting editing of the upstream site concomitantly decreases RNA editing of the second site 50-70 nucleotides downstream. This result suggests that supporting cis-/trans-interactions can be effective over larger distances and can affect more than one editing event.     

In vitro RNA editing in plant mitochondria does not require added energy

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.     

Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.     

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria: In search of the editosome

The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a ∼1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze–thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or “editosome.”     

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing

The genes encoding pea and potato mitochondrial tRNA^Gly and pea mitochondrial tRNA^Ser(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNA^Gly gene sequences revealed A₇:C₆₆ mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNA^Gly cDNA clones showed that these mispairings are not corrected by C₆₆ to U₆₆ conversions, as observed in plant mitochondrial tRNA^Phe. Likewise, a U₆:C₆₇ mismatch identified in the acceptor stem of the pea tRNA^Ser(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNA^Cys. In vitro processing reactions with the respective tRNA^Gly and tRNA^Ser(GCU) precursors show that such conversions are not necessary for 5′ and 3′ end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters. Received: 18 July 1997 / Accepted: 3 November 1997     

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.