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.     

RNA sequence and base pairing effects on insertion editing in Trypanosoma brucei

RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

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.     

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.     

Trypanosome RNA editing: simple guide RNA features enhance U deletion 100-fold

Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3' OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.     

Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification.

Chimeric RNA molecules were detected by polymerase chain reaction amplification of kinetoplast RNA using a 3' primer specific to mRNA and a 5' primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3' oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications result in the transfer of uridine residues from the gRNA 3' oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine "guide" nucleotides in the gRNA.     

Resolution of the RNA editing gRNA-directed endonuclease from two other endonucleases of Trypanosoma brucei mitochondria.

RNA editing in kinetoplastids, the specific insertion and deletion of U residues, requires endonuclease cleavage of the pre-mRNA at each cycle of insertion/deletion. We have resolved three endoribonuclease activities from Trypanosoma brucei mitochondrial extracts that cleave CYb pre-mRNA specifically. One of these, which sediments at approximately 20S and is not affected substantially by DTT, has all the features of the editing endonuclease. It cleaves CYb pre-edited or partially edited mRNA only when annealed to the anchor region of a cognate guide RNA (gRNA), and it cleaves accurately just 5' of the duplex region. Its specificity is for the 5' end of extended duplex RNA regions, and this prevents cleavage of the gRNA or other positions in the mRNA. This gRNA-directed nuclease is evidently the same activity that functions in A6 pre-mRNA editing. However, it is distinct and separable from a previously observed DTT-requiring endonuclease that sediments similarly under certain conditions, but does not cleave precisely at the first editing site in either the presence or absence of a gRNA. The editing nuclease is also distinct from a DTT-inhibited endonuclease that cleaves numerous free pre-mRNAs at a common structure in the region of the first editing site.     

Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata.

We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.     

A novel member of the RNase D exoribonuclease family functions in mitochondrial guide RNA metabolism in Trypanosoma brucei

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3' adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3'-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3' to 5' exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.     

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.     

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.     

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.