(Received for publication, October 30, 1995; and in revised form, December 14, 1995)
From the
RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.
The term RNA editing describes a variety of processing events
that generate RNAs that are different from those predicted by their
genes (reviewed in (1) ). In trypanosomatids (Trypanosoma, Herpetomonas, Crithidia, and Leishmania) and other related kinetoplastids (Trypanoplasma), RNA editing refers to the specific insertion
and deletion of uridylate (U) ()residues in mitochondrial
mRNAs. Since its discovery in kinetoplastids(2) , RNA editing
has been shown to create translational start and stop codons, correct
frameshifts, and extend open reading frames (reviewed in (3, 4, 5, 6) ). The extent of
editing can be small (e.g. the insertion of four U residues
into Trypanosoma brucei cytochrome oxidase subunit 2 (COII)
mRNA(2) ) or extensive (e.g. the creation of >50%
of the total transcript length of T. brucei COIII mRNA (7) ). Because trypanosomatids diverged from one another
200-300 million years ago(8, 9) , this RNA
editing is at least that old.
The genetic information directing RNA
editing resides in short (50-70 nt) mitochondrial
transcripts(10) . These guide RNAs (gRNAs) contain a 5` anchor
sequence (4-18 nt) that is complementary to the pre-edited mRNA
immediately 3` of the segment to be edited, a guiding region that is
complementary (allowing for G:U base pairing) to the fully edited mRNA
segment and a nonencoded (5-24 nt) 3` poly(U) tail (4) . Presumably the gRNA base pairs with the pre-edited mRNA
and directs editing to progress 5` from the anchor region, inserting
and deleting U residues to yield mRNA complementary to the guiding
region(10) . The poly(U) tail may further stabilize the duplex
of gRNA with the purine-rich mRNA (11) and/or serve as the
source of U residues for insertion(12) .
Blum et al. (10) proposed that each cycle of U insertion or removal involves an endonuclease to cleave the RNA, a terminal uridylyl transferase to insert or an exonuclease to delete U residues, and an RNA ligase to rejoin the 5` and 3` half mRNAs. Indeed, these proposed enzymatic activities are present in trypanosome mitochondria (13, 14, 15, 16) . Editing was later proposed to involve sets of transesterification reactions(12, 17) . In the most popular of these schemes, the 3` hydroxyl of the U tail of the gRNA attacks the mRNA at the first gRNA:mRNA mismatch, creating a gRNA-mRNA chimeric molecule. The chimera is then attacked by the 3` hydroxyl of the 5` mRNA fragment again adjacent to the duplex region, within the poly(U) tract, generating a partially edited mRNA and liberating the gRNA. Although the transesterification model has been considered very appealing because of its common mechanism with RNA intron removal (reviewed in (18) ) and because of the in vivo existence of gRNA-mRNA chimeric molecules in trypanosomes(12) , identical chimeric intermediates and partially edited RNAs could alternatively be generated by endonuclease cleavages and RNA ligations ((19) ; see Fig. 1).
Figure 1: One complete cycle of CYb RNA editing. A model for U insertional editing mediated by endonuclease plus RNA ligase involving a gRNA-mRNA chimeric intermediate is illustrated. In the first half cycle of editing, the extract endonuclease (or MBN) cleaves CYb pre-edited mRNA within the 3` editing domain, and the extract ligase (or T4 RNA ligase) covalently joins the 3` oligo(U) end of a CYb gRNA to the 3` half mRNA, generating a chimera with U residues at the junction. In the second half cycle, the extract endonuclease (or MBN) cleaves the chimera within the oligo(U) tract, and extract ligase (or T4 RNA ligase) then covalently rejoins the mRNA halves, generating a partially edited mRNA with U residues transferred from the gRNA tail to an editing site of the mRNA. The white boxes represent the editing domain of the mRNA, and the black boxes represent the oligo(U) tail of the gRNA.
In vitro studies on possible mechanisms of RNA editing have identified an editing domain-specific endonuclease in T. brucei that is single strand-specific (20) and cleaves cytochrome b (CYb) pre-edited mRNA predominantly at editing sites (ES) 2 and 3(14) . T. brucei mitochondrial extract was also shown to direct chimera formation, specifically joining a model CYb gRNA to pre-edited mRNA(21) , although the precise position of the junction (whether at ES1, ES2, and/or ES3) remained unclear. Investigations of the in vitro activity that forms chimeras have shown it to co-sediment with both RNA ligase (15) and the editing domain specific endonuclease(22) , demonstrating the feasibility of chimera formation through the enzymatic pathway(22) . Ruschéet al. (23) and Sabatini and Hajduk (24) then demonstrated that gRNA-mRNA chimeras are formed in trypanosome mitochondrial extract by a pathway that involves RNA ligase. Furthermore, data in Ruschéet al.(23) , in conjunction with results in this paper (see Fig. 2), demonstrate that the in vitro chimera formation pathway also utilizes the editing site-specific endonuclease of the extract. However, it remained unresolved whether the resultant chimeras could be intermediates in a complete cycle of editing.
Figure 2:
The
junction sequences of CYb gRNA-mRNA chimeras. A, CYb
pre-edited mRNA (0.5 pmol) and P-labeled CYb gRNA (1 pmol)
were incubated with mitochondrial extract (lane 2) or with MBN
and T4 RNA ligase (lane 3). The arrow indicates the
chimeric product. The
P-labeled gRNAs are off the bottom
of this photograph. The sizing markers used in this and subsequent
figures are a HpaII digest of pBR322 (lane 1). B, the gRNA-mRNA junction sequences from 37 cDNA clones of
chimeras generated using mitochondrial extract are shown, grouped
according to their mRNA junction sites. N is the nonediting
site immediately upstream of ES3, and the bold T represents 0,
1, or 2 T residues. Arrows show the locations of ES1, ES2, and
ES3. C, the gRNA-mRNA junction sequences from 34 cDNA clones
of chimeras generated using MBN and T4 RNA ligase are shown. Because
the clones of C derived from PCR amplification of
10
times more chimeric RNA than was used in the amplifications of B, it is likely that each clone represents a separate original
RNA molecule. The 3` ends of the gRNAs used in A had 1-3
U residues, whereas those in B and C had 3 U
residues.
In this study we report a full cycle of T. brucei U insertional editing, using CYb substrates and mitochondrial extract, as predicted by the enzymatic model involving a gRNA-mRNA chimeric intermediate (Fig. 1). The editing pattern of the resultant CYb partially edited mRNAs is similar to that of CYb partially edited RNAs generated in vivo. Furthermore, the utilization of ATP analogs indicates that previously reported U deletion (25) and U insertion (26) activities, like the above described activities, all involve RNA ligase.
CYb partially edited mRNAs were formed using the 5` half of CYb pre-edited mRNA (isolated from MBN-digested 5` end-labeled CYb pre-edited mRNA), the 3` half of CML15-2 RNA (prepared by digestion of pCp-labeled CML15-2 RNA with either mitochondrial extract or MBN), and 2 µl of mitochondrial extract or 15 U T4 RNA ligase as described for chimera formation. The samples were phenol-chloroform extracted, ethanol precipitated, electrophoresed, and visualized by autoradiography.
Chimeras can also be generated using MBN and T4 RNA ligase(20) . Their junctions should be at the same sites as those generated using mitochondrial extract because MBN also cleaves CYb pre-edited mRNA at ES2 and ES3(14) . As expected, in all 34 sequenced MBN/T4 RNA ligase-generated CYb chimera clones examined, the gRNA is joined to the CYb mRNA at either ES2 or ES3 (Fig. 2C). Obtaining the same profile of mRNA junction sites in CYb chimeras generated using either mitochondrial extract or a heterologous single strand-specific endonuclease plus RNA ligase provides additional support that the chimeras generated in the mitochondrial extract are formed by the concerted action of the extract endonuclease and RNA ligase.
Unlike the heterologous enzyme-generated CYb chimeras (Fig. 2C), most of the extract-generated chimeras did not retain all three U residues from the 3` tail of the gRNA (Fig. 2B). This observation explains the small size difference of the extract-generated and MBN/T4 RNA ligase-generated chimeras (Fig. 2A). Furthermore, chimeras generated in mitochondrial extract using a CYb gRNA with a 3` tail of 15 U residues retained 0-6 U residues at the gRNA-mRNA junction, and the 3` U tail of the free gRNAs was also observed to decrease in length (data not shown). These results suggest that a 3` exonuclease is active in T. brucei mitochondrial extracts. The observation that this 3`-terminal nucleotide removal appears to stop at the end of the oligo(U) tail (Fig. 2B) suggests that it may be a U-specific exonuclease. An analogous loss of U residues observed in the Leishmania tarentolae NADH dehydrogenase subunit 7 chimera-forming system (31) suggests that a similar exonuclease activity may be active in other kinetoplastids. In vivo, such a U-specific exonuclease activity could function in deleting genomically encoded U residues (10) or in removing excess U residues that were transferred to an editing site.
Figure 3:
Endonuclease specifically cleaves CYb
gRNA-mRNA chimeras. A, endonuclease cleavage reactions
contained 0.2 pmol of pCp-labeled CYb pre-edited mRNA (lanes
1-5) or 0.2 pmol of pCp-labeled CML15-2 RNA (lanes
7-9). (CML15-2 is chimeric RNA transcribed in
vitro from a reverse transcriptase-PCR amplified and cloned CYb
chimera that was generated by MBN and RNA ligase using a CYb gRNA with
a 3` tail of 15 U residues.) The reactions were incubated without
additions (lanes 1 and 7) or with 2 µl of
mitochondrial extract (lanes 2 and 8), 25 units of
MBN (lanes 3 and 9), RNase T1 (lane 4), or
RNase U2 (lane 5). The reactions of lanes 2 and 8 also contained 50 mg/ml of heparin. The bracket denotes
the 15 U residues of CML15-2 RNA that originally derived from the
gRNA tail. B, the predicted secondary structure for
CML15-2 mRNA (G = -40.5 kcal/mol) was
determined by the FOLD algorithm(30) . The major cleavage sites
for mitochondrial endonuclease (solid arrows) and for MBN (open arrows) are within the gRNA-derived U tract (bold
line). The first 29 nt and last 18 nt of this RNA are vector
sequences.
Figure 4:
Generation of CYb partially edited mRNA in vitro. A, pCp-labeled CML15-2 RNA was
cleaved using MBN (lane 3) or extract (lane 4) and
the isolated 3` half mRNAs (25 fmol) were mixed with P-labeled 5` half of CYb pre-edited mRNA (25 fmol,
MBN-derived) and treated with either 15 units of T4 RNA ligase (lane 3) or 2 µl of mitochondrial extract (lane
4). The arrow denotes partially edited mRNAs. 3` CML
indicates the 3` cleavage product of CML15-2 RNA; 5` pre
indicates the 5` cleavage product of CYb pre-edited RNA. Markers are
CYb pre-edited mRNA (lane 2) and HpaII cleaved pBR322 (lane 1). Lane 4 was exposed
4 times longer than lane 3. B, the sequences of cDNAs derived from
extract-generated (solid bars) and MBN/T4 RNA ligase-generated (stipled bars) CYb partially edited mRNAs, as in A,
are represented. The downstream sequence corresponds to CYb mRNA up to
ES2, at which point the number of inserted U residues and frequency of
representation are shown in the histogram. The upstream sequence also
corresponds to CYb mRNA, beginning at ES2 in most clones but at ES3 in
a small fraction of clones, including that used in Fig. 5.
Figure 5:
Endonuclease specifically cleaves CYb
partially edited mRNA. A, 0.2 pmol of pCp-labeled CYb
pre-edited mRNA (lanes 2-4) or 0.2 pmol of pCp-labeled
PE5-2 mRNA (lanes 5-8) were incubated without additions (lanes 2 and 5) or with 2 µl of mitochondrial
extract in the presence of 0.5 mg/ml heparin (lanes 3 and 6), 25 units of MBN (lanes 4 and 7), RNase
T1 (lane 8), or RNase U2 (lane 9). The 5 U residues
transferred to ES2 in PE5-2 are indicated by the bracket. B, analysis of extract cleavage sites using 3` labeled PE5-2 (left) or 5` labeled PE5-2 (right) is shown on a gel
that was run approximately twice as far as that of A. C, the secondary structure for PE5-2 mRNA (G = -41.1 kcal/mol) predicted by the FOLD algorithm (30) is illustrated. The 5 transferred U residues are indicated
by the bold line, and the arrows indicate the
endocnuclease cleavage sites.
To address whether the structure of the
CYb chimeric RNA determines its cleavage pattern, pCp-labeled
CML15-2 RNA was incubated with MBN, a probe for single-stranded
structure. This RNA was also specifically cleaved by MBN (Fig. 3A, lane 9), generating a family of 3`
mRNA products containing 10-13 U residues at their 5` ends.
This MBN cleavage within the poly(U) tract of CML15-2 RNA
indicates that this region of the chimera is single-stranded, a result
also predicted by analysis of this RNA using the FOLD algorithm (30) (Fig. 3B).
Such a stem-loop structure may well be a general feature of gRNA-mRNA chimeras, because the duplex involving the anchor region of the gRNA and its complement from the mRNA region presumably remains base paired, whereas the segment between those intervening regions shows only limited self-complementarity. Thus, preferential cleavage by single strand-specific nuclease in the oligo(U) region may be a general feature of chimeric molecules. The predicted structures for 20 trypansome pre-edited mRNA sequences also suggested a single strand-specific nuclease sensitivity in the region where editing begins (20) . Therefore, a common mitochondrial single strand-specific activity could recognize both the pre-edited mRNA and the gRNA-mRNA chimera, based on their secondary structures.
To verify the identity of these putative partially edited mRNAs, both the extract-generated and the T4 RNA ligase-generated products were reverse transcriptase-PCR amplified, subcloned, and sequenced. Partially edited mRNAs were indeed obtained (Fig. 4B). The majority of extract-generated partially edited mRNAs contained 8-9 U residues at ES2, whereas the majority of MBN plus T4 RNA ligase-generated mRNAs contained 10-11 U residues at ES2. These partially edited mRNAs represent products of a complete cycle of in vitro editing, demonstrating that it is indeed possible to transfer U residues from a gRNA tail, through a chimeric intermediate, and to an editing site within pre-edited mRNA. The fact that a full round of U insertional editing can also be mimicked by the structure-specific heterologous enzymes suggests that RNA secondary structural features direct the reaction. These results (Fig. 4B) also confirm the cleavage site data of Fig. 3A.
It is notable that this PE5-2 RNA as
well as CML15-2 RNA and CYb pre-edited mRNA is cleaved by the
extract endonuclease predominantly 2 nt from the 3` end of the
loop, whereas they are cleaved by MBN predominantly at the central
positions of the single-stranded loop. A cleavage preference of
mitochondrial endonuclease for a site a fixed distance from the 3` end
of a single-stranded loop might be predicted for an endonuclease
involved in editing.
Figure 6: Partially edited CYb mRNAs generated in vivo. Partially edited CYb mRNAs from in vivo T. brucei cellular RNA were cloned and sequenced(32) . Their sequences are grouped according to whether editing begins at ES2, at ES3, immediately 5` of ES3 (ES3`), or at ES1. The unedited sites of the editing domain 3` of the partially edited region are boxed. The unedited and fully edited CYb mRNA sequences are shown at the top with the editing sites numbered and the edited region shaded.
There are other examples in the literature of in vivo edited RNAs in which the editing also begins at ES2 rather than ES1. These include half of the reported Crithidia fasciculata MURF2 partially edited RNAs (33) and an additional T. brucei CYb RNA(27) . The literature also contains numerous examples of cDNAs containing an unedited site or sites within a larger otherwise correctly edited region (e.g. CYb of Trypanoplasma borrelli(34) , NADH dehydrogenase subunit 7 of T. brucei(35) , and G6(36) , CYb, and COIII of L. tarentolae(37) ), suggesting that editing in vivo may also permit a more relaxed versus a strict 3`-5` propagation.
Available data also imply that
RNA ligase is critical for the T. brucei A6 U deletion (25) and the L. tarentolae CYb U
insertion(26) , as it is for the T. brucei CYb chimera
formation (23, 24) and the U insertion examined here.
All of these activities are ATP-dependent, and all are unable to
utilize AMP-CPP (an ATP analog with a nonhydrolyzable -
linkage) (23, 24, 25, 26) , like RNA
ligases in general(38) . Although the report that the U
deletion activity does not utilize AMP-PCP (an ATP analog with a
nonhydrolyzable
-
linkage) suggested that U deletion may also
require
-
bond hydrolysis(25) , AMP-PCP also does not
efficiently support L. tarentolae CYb U
insertion(26) , T. brucei chimera-forming action (Fig. 7B), T. brucei RNA ligase activity (Fig. 7B), or even T4 RNA ligase activity (Fig. 7B). However, the L. tarentolae U
insertion, T. brucei chimera formation, and T. brucei RNA ligase activities all function with AMP-PNP (another ATP
analog with a nonhydrolyzable
-
linkage) ( (23) and (26) and Fig. 7), and therefore none of these reactions
require
-
bond hydrolysis. (This difference in analog
utilization presumably reflects the known structural differences
between ATP or AMP-PNP and AMP-PCP(39) .) S. Seiwert has kindly
assayed the T. brucei U deletion activity in the presence of
AMP-PNP and also found it to be active, (
)demonstrating that
this reaction also does not require
-
bond hydrolysis. Thus,
the T. brucei A6 U deletion activity and the L. tarentolae CYb U insertion activity, like the T. brucei CYb chimera
formation that can lead to U insertion, all show the same specificity
for various ATP analogs as do T. brucei and T4 RNA ligases (Fig. 7A). These activities all require
-
bond hydrolysis but not
-
bond hydrolysis and use AMP-PCP
inefficiently. The above data suggest that all these in vitro editing-like activities utilize some form of endonuclease- and RNA
ligase-based mechanisms.
Figure 7:
Utilization of ATP analogs by editing-like
activities. A, mitochondrial extracts were depleted of ATP
using hexokinase (23) and tested for their ability to support
chimera formation (top panel) or RNA ligation (middle
panel) in the absence of exogenously added ATP (lane 1)
or in the presence of ATP (lane 2) or the indicated ATP
analogs (lanes 3-5). The added ATP, but not the AMP-PNP,
is a substrate for the residual hexokinase(23) . T4 RNA ligase (bottom panel) was also tested for ligation under the above
conditions. B, the ability of ATP and ATP analogs to support
the in vitro T. brucei A6 U deletion(25) , L.
tarentolae CYb U insertion(26) , and T. brucei CYb chimera formation and RNA ligase activity (23, 24) are summarized in the white boxes.
The data in the shaded boxes were determined in this study,
and the box with the asterisk indicates unpublished
data kindly determined for us by S. Seiwert and reproduced
in our laboratory.