RNA Sequence and Base Pairing Effects on Insertion Editing in Trypanosoma brucei

Abstract

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.In kinetoplastid RNA editing, uridylates (U's) are inserted and removed at specific sites in mitochondrial (mt) premRNAs by a series of enzymatic steps to form mature, functional mRNAs (for recent reviews, see references 13, 17, and 37). Small guide RNAs (gRNAs) specify the edited sequence, which is complementary to the gRNAs by G · U and WatsonCrick base pairing (4). Editing is initiated by an endoribonucleolytic cleavage of the pre-mRNA at the editing site (ES) that is 5Ј to the anchor duplex between the 5Ј region of gRNA and the pre-mRNA downstream of the sequence that becomes edited. The U's are added or removed at the 3Ј end of the 5Ј pre-mRNA fragment that results from the cleavage (10,18,34).A 3Ј-terminal uridylyl transferase (TUTase) adds the U's, while a U-specific 3Ј exonuclease (exoUase) removes U's (2, 10). An RNA ligase rejoins the cleavage fragments after the U addition and removal. Overall, editing extends the gRNA/premRNA duplex in the 5Ј direction. The enzymatic activities that are required for editing are contained within a large multiprotein complex (1, 9, 29, 31), which sediments in glycerol gradients at 20S (9, 31) and is approximately 1,600 kDa in size (27).Progress has been made in elucidating how the edited sequence is specified by gRNA, but many questions remain unanswered. The cleavage step contributes to the specificity of editing by selecting the ES (4, 10, 18, 34). In deletion editing, this cleavage occurs downstream of the U's that will be removed (10, 34), and they are removed by the U-specific 3Ј exonuclease until the first non-U nucleotide is encountered (2, 11, 16a, 20a). Hence, selection of the ES by the endonuclease and the U specificity of the exonuclease may be more important than the sequence upstream of the ES for accurate editing at a deletion site.In insertion editing, cleavage occurs at an ES where the 3Ј nucleotide of the 5Ј fragment can base pair with the gRNA nucleotide adjacent to the purines t...

Identification of specific inhibitors for a trypanosomatid RNA editing reaction

“…3, +DMSO): Two guided U's are added to the 39 end of the linearized molecule, and the ligase activity of the complex re-forms the circular RNA with the two gRNAdirected U-insertions at the editing site. This is consistent both with an earlier assay exploiting a cleaved substrate (Igo et al 2002) and with the currently accepted reaction mechanism.…”

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

“…Formation of the edited product was severely diminished, indicating that it may play a role in tethering the 5' mRNA cleavage product during the editing reaction. Subsequent in vitro and gene knockdown experiments have lent credence to the importance of an upstream duplex and the U-tail in the editing reaction [27][28][29]. However, whether its role is limited to the tethering of the 5' cleavage product is unclear.…”

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

“…In Vitro Editing Assay. The following chemically synthesized RNAs (Integrated DNA Technologies, Coralville, IA) were used for the U-deletion and U-insertion in vitro editing assays (20,23): Udeletion, mRNA 5Ј-GAGAAGAAAGGGAAAGUUGU-GAUUUUGGAGUUAUAG-3Ј or 5Ј-UAGGGGGAGGA-GAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUU-AUAG-3Ј, gRNA 5Ј-GGAUAUAUACUAUAACUCCAC-CCUAUA ACUUUCCC-3Ј (20); U-insertion, mRNA 5Ј-UAGGGGGAGGAGAGAAGAAAGGGAAAGUACU-GAUUGGAGUUAUAG-3Ј, gRNA 5Ј-GGAUAUACUA-UAACUCCAAUAAACGAAGUUUUCCCUUUCUUU-3Ј. The peak L-complex gradient fractions of T. brucei mitochondrial lysate or TAP-purified L-complex, rTbMP90-CBP, and rLmMP90-CBP were incubated with synthetic RNAs.…”