Guide RNA-directed uridine insertion RNA editing in vitro.

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.     

Kinetoplastid RNA editing does not require the terminal 3' hydroxyl of guide RNA, but modifications to the guide RNA terminus can inhibit in vitro U insertion.

During RNA editing in kinetoplastid parasites, trans-acting guide RNAs (gRNAs) direct the insertion and deletion of U residues at precise sites in mitochondrial pre-mRNAs. We show here that some modifications to the 3' terminal ribose of gRNA inhibit its ability to direct in vitro U insertion. However, we found that gRNAs lacking this moiety in some circumstances support in vitro editing. Thus, the 3' OH is not required. Inhibition resulting from gRNA modification can be overcome by increasing the gRNA-pre-mRNA base-pairing potential upstream of the editing site, suggesting an importance for this interaction to productive processing.     

Kinetoplastid RNA editing: in vitro formation of cytochrome b gRNA-mRNA chimeras from synthetic substrate RNAs.

RNA editing in the kinetoplastid Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. The site and number of uridines added appears to be directed by small (approximately 70 nt) guide RNAs (gRNAs), which can base pair to the edited sequences. We examined reactions involving synthetic cytochrome b (CYb) gRNA and pre-edited mRNA in vitro. A major product of the in vitro reaction is a chimeric RNA molecule containing both gRNA and mRNA sequences. Formation of the CYb gRNA-mRNA chimera was specific, since such molecules did not accumulate when either the gRNA or mRNA was substituted with control RNAs. The reaction required a free 3' hydroxyl on the gRNA and was unaffected by capping of the gRNA's 5' end. Direct RNA sequencing indicated that the CYb gRNA is covalently linked via its 3' poly(U) tail to one of the editing sites on the CYb mRNA. These results suggest that the U's added during editing are donated by the poly(U) tail of a gRNA via a chimeric gRNA-mRNA intermediate.     

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity

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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.”     

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.     

In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNA(Trp)

Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNA(Trp) represents an outstanding specimen able to guide in cis, instead of in trans, two 2'-O-methylations in the pre-tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge-helix-bulge (BHB) splicing motif, while the RNA-guided methylation and pre-tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre-tRNA and tested its site-specific methylation activity. Recognition by L7Ae of hallmark K-turns at the C/D and C'/D' motifs appears as a crucial assembly step required for subsequent binding of a Nop5p-aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p-aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis-acting system to form guide RNA duplex(es) relative to classical trans- acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non-canonical stem of the K-turn, suggest that binding of Nop5p-aFib to the pre-tRNA-L7Ae complex might direct transition from a splicing-competent structure to an RNA conformer displaying the guide RNA duplexes required for site-specific methylation.     

RNA editing.

Since its discovery, RNA editing in kinetoplastid mitochondria has proven a fascinating topic of study, and the last one and a half years have witnessed enormous advances in our understanding of this unprecedented form of RNA processing. The information flow in this RNA editing, once considered a candidate for defying the central dogma, is now known to conform to the DNA-to-RNA-to-protein paradigm, with the novel feature that the sequence of an edited region is not actually present in any DNA segment, but instead derives by a novel micro-interdigitating of information encoded in multiple DNA regions.     

Kinetoplast DNA minicircles encode guide RNAs for editing of cytochrome oxidase subunit III mRNA.

Guide RNAs (gRNAs) for the editing of sites 1-8 of COIII mRNA and an "unexpected" partially edited COIII mRNA are encoded in the variable regions of specific kinetoplast DNA minicircles. The gRNAs can form 37 and 44 nucleotide perfect hybrids (allowing for G-U base pairs) with edited mRNAs. The gRNAs were detected on Northern blots and shown to have unique 5' ends situated close to the beginning of the potential base pairing with the edited mRNAs. We suggest that kinetoplast DNA minicircle molecules in general may encode gRNAs for editing of cryptogene mRNAs by a mechanism similar to that previously proposed for editing by maxicircle-encoded gRNAs.     

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

Recent advances in in vitrosystems 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 vitrosystems 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 vitroevidence 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.