Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae

Abstract

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

FIGURE 1.

Selection–amplification of RNAs supporting the insertion of Us within an in vitro editing extract. (A) A schematic illustrating one cycle of the selection–amplification strategy. A 70-nt RNA containing a 23-nt random region was circularized to block a 3′ U addition activity that is present within the editing extract. After treating the circular RNA with the editing extract, slower mobility RNAs were resolved from unedited RNA by gel electrophoresis. The RNA eluted from the excised part of the gel was amplified for T7 RNA polymerase mediated transcription by RT-PCR using the indicated primers. (B) The starting random RNA and the RNA from the first four cycles of selection were treated with the L. tarentolae editing extract. Slower migrating RNA, consistent with edited product, was visible after the fourth cycle of selection.

FIGURE 1.

Selection–amplification of RNAs supporting the insertion of Us within an in vitro editing extract. (A) A schematic illustrating one cycle of the selection–amplification strategy. A 70-nt RNA containing a 23-nt random region was circularized to block a 3′ U addition activity that is present within the editing extract. After treating the circular RNA with the editing extract, slower mobility RNAs were resolved from unedited RNA by gel electrophoresis. The RNA eluted from the excised part of the gel was amplified for T7 RNA polymerase mediated transcription by RT-PCR using the indicated primers. (B) The starting random RNA and the RNA from the first four cycles of selection were treated with the L. tarentolae editing extract. Slower migrating RNA, consistent with edited product, was visible after the fourth cycle of selection.

FIGURE 2.

Six of the selected RNAs have a highly similar sequence and can form secondary structures suggesting that the selected sequence functions as a cis-acting gRNA. (A) The RNA from the fourth cycle of selection was amplified by RT-PCR for cloning and sequencing. Six of the selected clones contain the indicated consensus sequence (red). Clone A-6 does not have the two 5′ As (parentheses) present on the other five clones. Selected sequence that is not part of the consensus is in green. (B) All six members of the consensus group have two Us inserted by the editing extract between A-19 and A-21 and can be folded into two alternative conformations in which two potential guiding nucleotides are placed either 5′ (conformation A) or 3′ (conformation B) of U-20. The sequence of the RNA that remained fixed during the selection is in black. A representative RNA, RNA A-1, was probed with DMS and CMCT under both native and denaturing conditions. The ratio of the intensity of the RT extension product obtained under native to that obtained under denaturing conditions was calculated at each indicated position. Mean ratios were calculated from four independent sets of DMS and three independent sets of CMCT reactions. A base is defined as protected if the native:denaturing ratio is <0.2, partially protected if the ratio is ≥0.2 and <0.5 and unprotected if the ratio is ≥0.5. (C) A representative gel.

FIGURE 2.

Six of the selected RNAs have a highly similar sequence and can form secondary structures suggesting that the selected sequence functions as a cis-acting gRNA. (A) The RNA from the fourth cycle of selection was amplified by RT-PCR for cloning and sequencing. Six of the selected clones contain the indicated consensus sequence (red). Clone A-6 does not have the two 5′ As (parentheses) present on the other five clones. Selected sequence that is not part of the consensus is in green. (B) All six members of the consensus group have two Us inserted by the editing extract between A-19 and A-21 and can be folded into two alternative conformations in which two potential guiding nucleotides are placed either 5′ (conformation A) or 3′ (conformation B) of U-20. The sequence of the RNA that remained fixed during the selection is in black. A representative RNA, RNA A-1, was probed with DMS and CMCT under both native and denaturing conditions. The ratio of the intensity of the RT extension product obtained under native to that obtained under denaturing conditions was calculated at each indicated position. Mean ratios were calculated from four independent sets of DMS and three independent sets of CMCT reactions. A base is defined as protected if the native:denaturing ratio is <0.2, partially protected if the ratio is ≥0.2 and <0.5 and unprotected if the ratio is ≥0.5. (C) A representative gel.

FIGURE 2.

Six of the selected RNAs have a highly similar sequence and can form secondary structures suggesting that the selected sequence functions as a cis-acting gRNA. (A) The RNA from the fourth cycle of selection was amplified by RT-PCR for cloning and sequencing. Six of the selected clones contain the indicated consensus sequence (red). Clone A-6 does not have the two 5′ As (parentheses) present on the other five clones. Selected sequence that is not part of the consensus is in green. (B) All six members of the consensus group have two Us inserted by the editing extract between A-19 and A-21 and can be folded into two alternative conformations in which two potential guiding nucleotides are placed either 5′ (conformation A) or 3′ (conformation B) of U-20. The sequence of the RNA that remained fixed during the selection is in black. A representative RNA, RNA A-1, was probed with DMS and CMCT under both native and denaturing conditions. The ratio of the intensity of the RT extension product obtained under native to that obtained under denaturing conditions was calculated at each indicated position. Mean ratios were calculated from four independent sets of DMS and three independent sets of CMCT reactions. A base is defined as protected if the native:denaturing ratio is <0.2, partially protected if the ratio is ≥0.2 and <0.5 and unprotected if the ratio is ≥0.5. (C) A representative gel.

FIGURE 3.

The selected sequence functions as a gRNA. (A) Changing the number of predicted guiding nucleotides resulted in a corresponding change in the number of Us inserted between A-19 and A-21. (B) Predicted pairing of the selected guiding sequence from clone A-1 with the starting randomer RNA used for the in vitro selection. The guiding sequence has an additional 5′ G to facilitate its in vitro transcription (Milligan and Uhlenbeck 1989). (C) The selected A-1 sequence can function as a trans-acting gRNA. A molar excess of an RNA containing nt G-30–G-50 of the selected A-1 sequence, including two guiding nucleotides, is able to direct correct editing of the starting random RNA population.

FIGURE 3.

The selected sequence functions as a gRNA. (A) Changing the number of predicted guiding nucleotides resulted in a corresponding change in the number of Us inserted between A-19 and A-21. (B) Predicted pairing of the selected guiding sequence from clone A-1 with the starting randomer RNA used for the in vitro selection. The guiding sequence has an additional 5′ G to facilitate its in vitro transcription (Milligan and Uhlenbeck 1989). (C) The selected A-1 sequence can function as a trans-acting gRNA. A molar excess of an RNA containing nt G-30–G-50 of the selected A-1 sequence, including two guiding nucleotides, is able to direct correct editing of the starting random RNA population.

FIGURE 3.

The selected sequence functions as a gRNA. (A) Changing the number of predicted guiding nucleotides resulted in a corresponding change in the number of Us inserted between A-19 and A-21. (B) Predicted pairing of the selected guiding sequence from clone A-1 with the starting randomer RNA used for the in vitro selection. The guiding sequence has an additional 5′ G to facilitate its in vitro transcription (Milligan and Uhlenbeck 1989). (C) The selected A-1 sequence can function as a trans-acting gRNA. A molar excess of an RNA containing nt G-30–G-50 of the selected A-1 sequence, including two guiding nucleotides, is able to direct correct editing of the starting random RNA population.

FIGURE 4.

U insertions occur both 5′ and 3′ of a genomically encoded U. (A) The selected RNA can potentially form two different conformations in which guiding nucleotides direct insertions either 5′ (conformation A) or 3′ (conformation B) of U-20. A TLC-based assay was used to distinguish between the two possibilities. Unlabeled circular A-1 RNA with one guiding nucleotide was treated with editing extract in the presence of [α-³²P]-UTP. Whereas the labeled phosphate will be inserted immediately 3′ of A-19 by editing of conformation A, it will be inserted immediately 3′ of U-20 by editing of conformation B. Complete alkaline hydrolysis of the gel-purified extract-treated circular RNA will produce a labeled 2′-3′ cyclic A or U monophosphate depending on whether the editing occurred 5′ or 3′ of U-20. (B) Analysis of the hydrolysis products on poly(ethylenimine) cellulose TLC plates resolved in 1 M formic acid indicates that U insertions occur equally both 5′ and 3′ of U-20. Unlabeled 3′ AMP and 3′ UMP standards were used as markers and were visualized under UV light. The TLC plate is representative of three independent experiments.

FIGURE 4.

U insertions occur both 5′ and 3′ of a genomically encoded U. (A) The selected RNA can potentially form two different conformations in which guiding nucleotides direct insertions either 5′ (conformation A) or 3′ (conformation B) of U-20. A TLC-based assay was used to distinguish between the two possibilities. Unlabeled circular A-1 RNA with one guiding nucleotide was treated with editing extract in the presence of [α-³²P]-UTP. Whereas the labeled phosphate will be inserted immediately 3′ of A-19 by editing of conformation A, it will be inserted immediately 3′ of U-20 by editing of conformation B. Complete alkaline hydrolysis of the gel-purified extract-treated circular RNA will produce a labeled 2′-3′ cyclic A or U monophosphate depending on whether the editing occurred 5′ or 3′ of U-20. (B) Analysis of the hydrolysis products on poly(ethylenimine) cellulose TLC plates resolved in 1 M formic acid indicates that U insertions occur equally both 5′ and 3′ of U-20. Unlabeled 3′ AMP and 3′ UMP standards were used as markers and were visualized under UV light. The TLC plate is representative of three independent experiments.

FIGURE 5.

A duplex immediately upstream of the editing site affects insertional editing. (A) The location of some of the mutations used to probe the significance of the duplex upstream of the editing sites (red). Only the structure of the parental RNA indicated in Figure 2B ▶ is supported by chemical modification studies. (B) Disruption of the duplex immediately upstream of the editing sites cannot be compensated by additional stabilization of the downstream duplex. The gel is representative of two sets of reactions. (C) Additional stabilization of the upstream duplex partially inhibits the editing reaction. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from two sets of reactions are indicated beneath the gel.

FIGURE 5.

A duplex immediately upstream of the editing site affects insertional editing. (A) The location of some of the mutations used to probe the significance of the duplex upstream of the editing sites (red). Only the structure of the parental RNA indicated in Figure 2B ▶ is supported by chemical modification studies. (B) Disruption of the duplex immediately upstream of the editing sites cannot be compensated by additional stabilization of the downstream duplex. The gel is representative of two sets of reactions. (C) Additional stabilization of the upstream duplex partially inhibits the editing reaction. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from two sets of reactions are indicated beneath the gel.

FIGURE 5.

A duplex immediately upstream of the editing site affects insertional editing. (A) The location of some of the mutations used to probe the significance of the duplex upstream of the editing sites (red). Only the structure of the parental RNA indicated in Figure 2B ▶ is supported by chemical modification studies. (B) Disruption of the duplex immediately upstream of the editing sites cannot be compensated by additional stabilization of the downstream duplex. The gel is representative of two sets of reactions. (C) Additional stabilization of the upstream duplex partially inhibits the editing reaction. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from two sets of reactions are indicated beneath the gel.

FIGURE 6.

Sequence specificity of the nucleotides immediately flanking the editing site. (A) Two base pairs immediately downstream from the editing site were randomized for one cycle of reselection. The sequences of the reselected edited RNAs are indicated. (B) The two indicated base pairs upstream of the editing site were also randomized for an independent reselection. (C) Editing of the parental A-1 RNA (UA/AU), the RNA randomized at the downstream (A) positions (NN/NN), and two RNAs from the downstream reselection that maintain Watson–Crick pairing within the selected sequence. The gel is representative of three sets of reactions. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from the three data sets are indicated beneath the gel. (D) Editing of the parental A-1 RNA (UA/AU) and four RNAs from the downstream reselection that have mismatches within the selected sequence. The gel is representative of two sets of reactions. (E) Editing of the parental A-1 RNA (GA/CU), the RNA randomized at the upstream (B) positions (NN/NN), and four RNAs obtained from the upstream reselection. The gel is representative of three sets of reactions.

FIGURE 6.

Sequence specificity of the nucleotides immediately flanking the editing site. (A) Two base pairs immediately downstream from the editing site were randomized for one cycle of reselection. The sequences of the reselected edited RNAs are indicated. (B) The two indicated base pairs upstream of the editing site were also randomized for an independent reselection. (C) Editing of the parental A-1 RNA (UA/AU), the RNA randomized at the downstream (A) positions (NN/NN), and two RNAs from the downstream reselection that maintain Watson–Crick pairing within the selected sequence. The gel is representative of three sets of reactions. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from the three data sets are indicated beneath the gel. (D) Editing of the parental A-1 RNA (UA/AU) and four RNAs from the downstream reselection that have mismatches within the selected sequence. The gel is representative of two sets of reactions. (E) Editing of the parental A-1 RNA (GA/CU), the RNA randomized at the upstream (B) positions (NN/NN), and four RNAs obtained from the upstream reselection. The gel is representative of three sets of reactions.

FIGURE 6.

Sequence specificity of the nucleotides immediately flanking the editing site. (A) Two base pairs immediately downstream from the editing site were randomized for one cycle of reselection. The sequences of the reselected edited RNAs are indicated. (B) The two indicated base pairs upstream of the editing site were also randomized for an independent reselection. (C) Editing of the parental A-1 RNA (UA/AU), the RNA randomized at the downstream (A) positions (NN/NN), and two RNAs from the downstream reselection that maintain Watson–Crick pairing within the selected sequence. The gel is representative of three sets of reactions. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from the three data sets are indicated beneath the gel. (D) Editing of the parental A-1 RNA (UA/AU) and four RNAs from the downstream reselection that have mismatches within the selected sequence. The gel is representative of two sets of reactions. (E) Editing of the parental A-1 RNA (GA/CU), the RNA randomized at the upstream (B) positions (NN/NN), and four RNAs obtained from the upstream reselection. The gel is representative of three sets of reactions.

FIGURE 6.

Sequence specificity of the nucleotides immediately flanking the editing site. (A) Two base pairs immediately downstream from the editing site were randomized for one cycle of reselection. The sequences of the reselected edited RNAs are indicated. (B) The two indicated base pairs upstream of the editing site were also randomized for an independent reselection. (C) Editing of the parental A-1 RNA (UA/AU), the RNA randomized at the downstream (A) positions (NN/NN), and two RNAs from the downstream reselection that maintain Watson–Crick pairing within the selected sequence. The gel is representative of three sets of reactions. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from the three data sets are indicated beneath the gel. (D) Editing of the parental A-1 RNA (UA/AU) and four RNAs from the downstream reselection that have mismatches within the selected sequence. The gel is representative of two sets of reactions. (E) Editing of the parental A-1 RNA (GA/CU), the RNA randomized at the upstream (B) positions (NN/NN), and four RNAs obtained from the upstream reselection. The gel is representative of three sets of reactions.

FIGURE 6.

Sequence specificity of the nucleotides immediately flanking the editing site. (A) Two base pairs immediately downstream from the editing site were randomized for one cycle of reselection. The sequences of the reselected edited RNAs are indicated. (B) The two indicated base pairs upstream of the editing site were also randomized for an independent reselection. (C) Editing of the parental A-1 RNA (UA/AU), the RNA randomized at the downstream (A) positions (NN/NN), and two RNAs from the downstream reselection that maintain Watson–Crick pairing within the selected sequence. The gel is representative of three sets of reactions. For each RNA, the fraction of correctly edited product was normalized to the fraction of correctly edited parental RNA. The mean relative editing values and standard deviations calculated from the three data sets are indicated beneath the gel. (D) Editing of the parental A-1 RNA (UA/AU) and four RNAs from the downstream reselection that have mismatches within the selected sequence. The gel is representative of two sets of reactions. (E) Editing of the parental A-1 RNA (GA/CU), the RNA randomized at the upstream (B) positions (NN/NN), and four RNAs obtained from the upstream reselection. The gel is representative of three sets of reactions.

FIGURE 7.

RNA editing increases linearly with added extract. (A) One picomole of radiolabeled RNA was incubated with the indicated amount of editing extract. The ratio of correctly edited product to total RNA, including correctly edited, unedited, incorrectly edited, and degradation products, was calculated for each reaction. Each point is the mean of three independent reactions and the standard deviation, where significant, is indicated by a vertical line. (B) A representative set of reactions.

FIGURE 7.

RNA editing increases linearly with added extract. (A) One picomole of radiolabeled RNA was incubated with the indicated amount of editing extract. The ratio of correctly edited product to total RNA, including correctly edited, unedited, incorrectly edited, and degradation products, was calculated for each reaction. Each point is the mean of three independent reactions and the standard deviation, where significant, is indicated by a vertical line. (B) A representative set of reactions.