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Previous Article | Next Article 
Molecular and Cellular Biology, March 2002, p. 1567-1576, Vol. 22, No. 5
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.5.1567-1576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Seattle Biomedical Research Institute, Seattle, Washington 98109,1 Department of Pathobiology, University of Washington, Seattle, Washington 981952
Received 16 July 2001/ Returned for modification 10 September 2001/ Accepted 29 November 2001
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 3'-terminal uridylyl transferase (TUTase) adds the U's, while a U-specific 3" 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/pre-mRNA duplex in the 5' 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 3' 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 3' nucleotide of the 5' fragment can base pair with the gRNA nucleotide adjacent to the purines that guide the U insertions. The U specificity of TUTase must play an important role in insertion editing, but gRNA/mRNA interactions upstream of the ES and the number of guiding purines also appear important to specificity in insertion editing (7, 16). The sequence of the gRNA appears to contribute to both latter sources of specificity (4, 8). The gRNA/pre-mRNA base pair immediately 5" to the ES may help specify the number of inserted U's. It may present the 5" fragment as a substrate for TUTase, help to stabilize the added U's by base pairing with the gRNA purines, or position the 5" fragment in register with gRNA so that the 5" fragment with the correct number of added U's is properly aligned for ligation. In addition, the strong bias for purine ribonucleotides upstream of insertion ESs (6) may reflect selection against base pairing with guiding nucleotides or adjacent purines in gRNA that do not specify insertion of U's.
We used the precleaved insertion editing system (16) to examine the effects of gRNA and pre-mRNA mutations on the U addition and ligation steps of editing to test the above possibilities. We used this system because it does not require prior cleavage and allows mRNA and gRNA sequence changes that would normally disrupt cleavage or change the location of the ES. Where possible, we used an uncleaved substrate in vitro to examine a full round of insertion editing to test the effects of these changes on endonucleolytic cleavage and on the series of editing steps. We found that sequence elements of gRNA and pre-mRNA in and around the ES contribute to specification of the edited mRNA sequence in insertion editing. We also found that no editing occurs in in vitro substrates with a pyrimidine that is upstream and immediately adjacent to the ES. Furthermore, a stable pre-mRNA/gRNA duplex upstream of the ES enhanced editing efficiency except when immediately adjacent to the editing site.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transcription templates for variants of 5'CL18 (Fig. 1A)
with altered 3' termini were prepared by PCR of 5'CL18-Tmp1 (16) using oligonucleotide primer EcoRI T7 (16) and variants of primer 5"CL18-3" (5"-NCTACGTCTCATACTTCCTATAG-3"), where N is complementary to the desired 3"-terminal nucleotide. Transcription templates for derivatives of gPCA6-1A with various guiding nucleotides (Fig. 1) were synthesized by PCR of gPCA6-3A-Tmp1 (16) with EcoRI T7 and primers gPCA6-N (5"-GGAAGTATGAGACGTAGGNATCGGAG-3"), where N is complementary to the desired guiding nucleotide. Similarly, templates for transcription of gPCA6-2A derivatives with two guiding nucleotide were synthesized by PCR of gPCA6-3A-Tmp1 with EcoRI T7 and primers of sequence 5"-GGAAGTATGAGACGTAGGN1N2ATCGGAG-3", where N1N2 is the complement of the two guiding nucleotides read 5"
3" with respect to gRNA (i.e., right to left in Fig. 3).
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3" with respect to gRNA (i.e., read right to left in Fig. 4B). Variants of gA6[14]USD-2A (16) with substitutions at position Xg, as shown in Fig. 7B, were prepared using 3" primers 5"-AAAGAAAGGGAAAACTTC NTTATTGGAGTT-3", where N is complementary to Xg.
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Precleaved substrate RNAs were labeled at the 5' terminus of the 5" cleavage fragment by alkaline phosphatase and polynucleotide kinase treatment, as described (16). Full-round editing substrates were labeled at the 3' terminus by ligation of [5"-32P]pCp (38).
Preparation of mt extract. Partially purified Trypanosoma brucei mt extract was prepared either by sequential SP Sepharose and Q Sepharose chromatography or by the same method followed by gel filtration on a Superose 6 column, as described (27), starting with mitochondria isolated from T. brucei strain IsTaR 1.7a (15) lysed in 0.7% (vol/vol) Triton X-100 (29). Fractions from the final step of these purification procedures were tested for insertion editing activity, and peak fractions were used for the experiments. Extract was stored in 10 to 15% (vol/vol) glycerol at -70°C between purification and use in editing assays. Both types of extract displayed the same editing activity, though fractions from the three-step purification contained less nonspecific RNase activity (data not shown).
Precleaved and full-round editing assays. Editing reactions, and the separation and visualization of the edited products, were performed as described by Igo et al. (16). Reactions performed in the absence of UTP were used as background measurements for quantifying U addition products in precleaved insertion reactions. Controls without UTP were also used for measuring background for quantifying full-round insertion editing, rather than without gRNA, because the stable gRNA-mRNA structure caused a small amount of retardation of input RNA during gel electrophoresis. Parallel time course assays were stopped by the addition of 2 µl of stop buffer (16) and incubation on ice prior to phenol-chloroform extraction of RNA. The abundance of products of the editing reaction was initially calculated as the percentage of total input RNA and then normalized to the abundance of the corresponding products in control reactions, except as indicated. Abundance of total U addition products was computed as the sum of ligated and nonligated products with the specified number of added U's. Unless otherwise specified, numerical data are reported as the mean of two independent experiments.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 2 shows that the initial rate of U addition with a single guiding C was similar to that with a guiding A, but that the reduced accumulation of +1 U addition product was reached quickly. This was also the case with a nonligatable 3' fragment which has no 5' phosphate, although the overall rate of addition was lowered, as reported previously (16), resulting in accumulation of +1 U addition product being reduced by about 35-fold (data not shown). These data suggest that the guiding nucleotide does not affect U addition but stabilizes the added U, perhaps by blocking U removal by the 3" exoUase that is present in the editing complex, as has been shown for purified exoUase (2).
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70% reduction in edited product, again indicating an effect on ligation. Replacement of one guiding A with a C or U reduced the amount of +2 U addition product, increased the amount of +1 U addition product, and generated some +3 U addition product. These replacements also substantially reduced the amounts of accurately edited RNA (i.e., containing two inserted U's) and produced prominent ligation products with one or no inserted U's. When the guiding A's were replaced with two C's, addition products with up to four U's were generated (Fig. 3). No edited RNA was produced in this case, but ligation products with no inserted U's were formed. The ligation products may reflect base pairing of the terminal G of the 5' fragment with the guiding C's (see Discussion). Replacement of the G with an A along with a compensatory change in the gRNA to retain base pairing still resulted in preferential production of the ligated product with no inserted U's, although a small amount of ligated RNA with one or two inserted U's was generated. Overall, these results indicate that U addition does not require a complementary nucleotide in the gRNA and thus that it is not templated, although base pairing appears to stabilize the added U, since the addition products continue to accumulate quickly after a short period of time only in the presence of a guiding purine (Fig. 2). In addition, base pairing of the added U's with the gRNA substantially enhances ligation.
The effects of pyrimidine guiding nucleotides were also pronounced when insertion editing was examined by the in vitro assay that requires cleavage of the substrate (16) (Fig. 4). Replacement of one of the three guiding A's in gA6[14]USD-3A (Fig. 4A) with a C resulted in cleavage at the same site as with the wild-type gRNA (ES2) except when the C was adjacent to the anchor duplex (Fig. 4B). In the latter case, cleavage occurred one or two nucleotides upstream of ES2, probably due to base pairing of the guiding nucleotides (lowercase in Fig. 4B) with the G or the G and U upstream of ES2 in A6AC. The resultant edited RNA reflected the number of guiding A's adjacent to the anchor duplex, and no edited RNA was produced when the replacing C shifted the cleavage site. Similar results were obtained using A6AC precleaved at ES2 (data not shown). Replacement of one of the three guiding A's with U rather than C resulted in some cleavage at ES2 but also some cleavages further upstream (Fig. 4C). In addition, a similar but low abundance of edited RNA with one, two, or three inserted U's was generated. These multiple cleavage sites and production of multiple edited RNAs may reflect the potential base pairing between the substituted U and the G or A that is on either side of ES2 in the mRNA.
Nucleotides and base pairing adjacent to the ES. The effects on in vitro editing of various nucleotides adjacent to the ES were examined because there is a nucleotide bias adjacent to the insertion ESs in pre-mRNAs, especially against C's immediately upstream of the ES (6). The efficiency and accuracy of insertion editing were tested for all possible Watson-Crick and G · U base pairs 5' to the ES using the precleaved system (Fig. 5). Conversion of the upstream G · C base pair to G · U (Xm = G in mRNA and Xg = U in gRNA, Fig. 5A) did not substantially affect the amount of resultant +2 U addition product or +2 edited RNA but did increase the amount of +1 addition product (Fig. 5B). Substitution of A · U for G · C resulted in a 70% increase in +2 U addition product and +2 edited RNA. In contrast, editing was significantly reduced when Xm was a pyrimidine. This effect was most pronounced with C in position Xm, where both the +2 addition product and edited RNA were reduced to less than 2% of the wild-type levels. In addition, the 3' terminal U was removed from a small fraction (1 to 2%) of the input RNA, probably by the U-specific exonuclease in the extract, suggesting that exoUase is more efficient than TUTase in this context (Igo et al., submitted for publication).
Precleaved substrate with the upstream U · A underwent insertion editing with about half the efficiency of the wild type, but only small amounts of U addition and edited products were observed with an upstream U · G base pair. The G in gRNA prevented U removal as well as did A. Formation of ligation product with no added U's was greater than the +2 edited product with an upstream pyrimidine (Xm in Fig. 5A). This may again reflect the tendency of a purine guiding nucleotide adjacent to the anchor duplex to bridge with the upstream nucleotide and enhance ligation. In contrast, a pyrimidine downstream of the ES (Zm in Fig. 5A) had little effect on precleaved editing, although a U at this position promoted ligation without U addition. These results may, in part, account for the observed bias against C's upstream of insertion ESs, but not the bias against pyrimidines downstream of the ES (6).
The effects on U addition and editing of gRNA/pre-mRNA base pairing adjacent to the ES were examined (Fig. 6). A single G · G mismatch upstream of the ES substantially reduced the production of +2 U addition product and edited RNA but retained the accuracy of precleaved insertion editing (Fig. 6B). Production of accurately edited RNA was reduced by over 90% with two or three G · G mismatches upstream and substantial +1 U addition product, and inaccurately edited RNA (with one U inserted) was generated with three mismatches. The pattern of U addition with three upstream G · G mismatches resembled that obtained in the absence of gRNA. One, two, or three mismatches downstream of the ES progressively diminished the production of edited RNA but, unlike upstream mismatches, did not greatly affect the accuracy of editing (Fig. 6B).
The prominent U addition product contained two U's, as specified by the gRNA. A small fraction of RNA with more than two added U's was produced, especially with three mismatches, perhaps reflecting base pairing of various added U's with the two A's in the gRNA. Overall, base pairing immediately upstream of the ES affects precleaved insertion editing more than similar changes downstream, perhaps reflecting a focus of the catalytic activity on the 3' end of the 5' fragment.
The effects of specific nucleotides and base pairing 5' to the ES on full-round insertion editing (including cleavage) at ES2 of A6 RNA were examined (Fig. 7). Editing was examined using A6-eES1/gA6[14] (18) (Fig. 7A) and A6AC/gA6[14]USD (16) (Fig. 7B) pre-mRNA/gRNA pairs in which the pre-mRNA G upstream of ES2 and the corresponding C in the gRNA were replaced with each of the other nucleotides and tested in all possible combinations. With the A6-eES1/gA6 pair, insertion editing was only detected in the presence of the wild-type G · C upstream base pair. However, cleavage and chimera formation occurred with a purine upstream of the ES regardless of the corresponding nucleotide in the gRNA, and no cleavage was observed with a C upstream of the ES (data not shown). A6-eES1 pre-mRNA with U upstream of the ES was not tested, since the U could base pair with the guiding A in gRNA. Accurate editing occurred when the wild-type G · C pair in ACA6/gA6[14]USD was replaced with an A · U base pair but was reduced by about 50% (Fig. 7B).
Editing also occurred, but at a lower level, when G · C was replaced with other base pairs, including G · U, and the +1 edited product was increased relative to the wild type. The amount of edited RNA was correlated with predicted base pair stability. Cleavage occurred at ES2 in each of these cases, but only in the presence of gRNA. Thus, cleavage, U addition, and ligation can all proceed without an upstream base pair, but this base pair greatly enhances accurate editing. However, no editing or cleavage at ES2 or nearby was observed when the upstream nucleotide was a pyrimidine (data not shown), perhaps because the U or C in the mRNA may base pair with the guiding A in gRNA and block substrate recognition by the endonuclease. Hence, the number of inserted U's was efficiently specified by the guiding A's only when a purine was present immediately upstream of the ES that could form a G · C or A · U base pair with the nucleotide 3' to the guiding A's.
A similar but more limited analysis was performed using CYb pre-mRNA. This analysis used gCYb[558]USD gRNA, modified from the wild-type gRNA (30), which does not support editing in vitro. gCYb[558]USD can form a duplex upstream of ES1, creating a 6-nt bulge in pre-mRNA (Fig. 8A). The major edited product contained two inserted U's, although considerable +1 U and some +3 edited RNA was also produced (Fig. 8B). Use of pre-mRNA with A substituted for the upstream G and gRNA with C substituted for the corresponding U to provide G · C, A · U, and A · C base pairs resulted in reduced editing and cleavage (Fig. 8B). In particular, there was very little cleavage or editing with the G · C base pair. However, precleaved editing of this substrate-gRNA pair was efficient (data not shown), and thus the presence of this G · C upstream base pair appears to specifically affect cleavage. Thus, wild-type A6 and CYb pre-mRNAs were most efficiently edited with wild-type gRNA, and base pairing upstream of the ES appears to be necessary but not sufficient for efficient, specific editing.
Stability and position of an upstream duplex. The effect of the location of an upstream duplex on insertion editing was examined, since a strong gRNA/pre-mRNA upstream duplex is known to increase the efficiency of in vitro editing (5, 11, 16, 19) (Fig. 9). Little cleavage or editing of A6AC pre-mRNA was observed with a 15-bp duplex immediately adjacent to ES2 (Fig. 9B). Otherwise, the efficiency of full-round insertion editing correlated with the proximity of the stable duplex to the ES, with duplexes nearer the ES promoting more editing, although production of the 3' cleavage product was similar. The most efficient editing was achieved using gA6[14]USD, which leaves 4 nt of single-stranded RNA upstream of ES2 upon binding to A6AC.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increased formation of aberrant editing products when gRNA contains guiding pyrimidines (Fig. 1 and 3) may result from base pairing of the guiding pyrimidine with the substrate purine that is next to the ES in either the 5' or 3' mRNA fragment, as illustrated in Fig. 10. Such base pairing would act to bulge out a nucleotide in the gRNA and would enhance ligation by bringing the mRNA termini together. These bulged structures are less energetically favorable than structures in which the helices both 5" and 3" of the ES are continuous, but thermodynamic considerations (35) suggest that they occur as a significant proportion of all editing intermediates. For example, if the guiding A in gPCA6-1A is replaced with a C (Fig. 1A and 1B, lane C), the 3"-terminal G of 5"CL18 can pair with the guiding C, bulging out a C in gRNA, as shown in Fig. 10A (left arrow). The G°37 of this alternate structure is estimated to be 4.2 kcal/mol, less favorable than the unbulged structure (35), but some of this difference is expected to be compensated for by the stacking interaction between the 3"-terminal G and the 5"-terminal A of the 3" fragment.
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U addition during precleaved insertion occurred opposite guiding C's (Fig. 3), and neither precleaved nor full-round insertion editing strictly required a potential base pair to secure the 3' end of the 5' fragment (Fig. 6 and 7). That this was not observed previously (18) is most likely due to the greater sensitivity of the editing assays used here. This U addition to the 5" fragment in the absence of base pairing is indicative of the terminal transferase rather than polymerase activity for the addition of U's during editing. The specificity of this activity for U's (N. Ernst, R. P. Igo, Jr., B. Panicucci, A. K. Panigrahi, and K. Stuart, unpublished data) contributes to accuracy of the editing. The gene for the editing 3" TUTase(s) has not yet been identified. The guiding purines appear to stabilize the retention of the correct number of U's that are added to the 5" fragment. This stabilization may entail protecting the added U's from removal by exoUase (7, 16, 23; R. P. Igo, Jr., et al., unpublished data) and/or enhancing ligation by bringing the 3"-terminal hydroxyl of the 5" mRNA fragment in close proximity to the 5"-terminal phosphate of the 3" mRNA fragment (3, 16). The slight reduction in abundance of U addition products with two guiding G's rather than A's (Fig. 3) suggests that less-stable base pairing renders the added U's more susceptible to exoUase activity and less well tethered for ligation.
The presence of base pairs and certain single nucleotides immediately upstream of the ES has a more profound effect on insertion editing than do specific nucleotides and base pairing downstream of the ES. Cleavage, U addition, and ligation all occur in the absence of an upstream base pair, but only in the presence of this base pair do all three activities act together to produce gRNA-specified sequence, as has been observed by others (11). Upstream mismatches greatly reduce precleaved editing to the extent that U addition with three mismatches resembles that obtained without gRNA (Fig. 6). Accurately edited RNA is the most prominent edited product in full-round in vitro editing only when there is an upstream base pair (Fig. 7 and 8) and the upstream base pair closest to the anchor duplex influences the number of inserted U's (Fig. 4), as can G · C, G · U, and even A · C base pairs, even with a strong potential upstream duplex (Fig. 1 and 2).
Ligation seems to be efficient only when the terminal nucleotide is paired with gRNA, and the efficiency is greater with more stable base pairing (Fig. 1 and 2). An upstream base pair is required but nevertheless is not sufficient for accurate insertion editing (Fig. 8). The anchor duplex is required for editing (10, 34; N. Ernst, unpublished results), and base pairing immediately downstream of the ES affects the site of cleavage and hence ES selection but not the number of inserted U's, although the efficiency of ligation is reduced (Fig. 6). These data suggest that the editing complex binds the anchor duplex but also interacts with RNA upstream of the ES.
The results reported here suggest that additional factors contribute to the rarity of C's immediately 5' to insertion ESs (6) beyond the possible selection against C's as a result of their potential base pairing with guiding G's or even A's. Such upstream C's impair both cleavage (data not shown) and U addition (Fig. 5), even in the absence of gRNA (N. Ernst et al., unpublished data). These data suggest that characteristics of the editing machinery itself contribute to the sequence bias. The lack of cleavage downstream of insertion site C's is surprising, given the bias for C's upstream of deletion sites (5). This may be due to mRNA/gRNA structure differences between insertion and deletion substrates. In addition, this may reflect the specificity of the editing endonuclease, or the editing complex may contain two (or more) endonucleases, each with a substrate specificity biased for insertion or deletion substrates.
In contrast to the upstream C, the presence of a pyrimidine 3' to the ES had little effect on precleaved insertion editing (Fig. 5). Addition at low frequency of one more U than specified may reflect breathing at the 5' end of the anchor duplex, which would permit base pairing of the additional U with the Zg purine, perhaps protecting it from removal by 3" exoUase. Thus, the sequence bias and effects of specific nucleotides 5" to the ES on editing activities further indicate the focus of these activities on the 3" end of the 5" cleavage fragment.
A stable upstream duplex that can tether the 5' fragment to gRNA increases insertion editing substantially (5, 11, 16, 19), although this duplex is not present in vivo. We found that the amount and fidelity of editing increased with duplex proximity to the ES, although no full-round editing occurs when the duplex is adjacent to the ES, since no endonucleolytic cleavage occurs (Fig. 9 and data not shown). A larger information region, as in the constructs here, can reduce editing (11). However, placement of an upstream duplex close to the ES in constructs in which the informational region size remains constant increases editing (5), revealing the effect of the upstream duplex.
The large reduction in editing when the upstream duplex abuts the ES reported here for full-round insertion editing and seen in deletion editing (11) is due to the lack of endonucleolytic cleavage. This suggests that the editing endonuclease(s) requires at least one unpaired nucleotides in the substrate. However, cleavage does not always occur immediately 5' to the anchor duplex, even when a bulge is present in pre-mRNA (Fig. 4) (S. D. Lawson, unpublished data). Thus, our understanding of the factors determining the cleavage site is still incomplete. These data suggest that, in vitro, the upstream duplex positions the 3' end of the 5" fragment close to the 3" TUTase and ligase active-site domains. The absence of such upstream duplexes in vivo implies that the 3" oligo(U) tail that is present on all gRNAs could play a comparable role by binding with purine-rich sequences upstream of the ES, and indeed it can bind close to the ES (21). Protein components of the editing complex may also play a role in positioning the 3" end of the 5" fragment, and some of these proteins may bind the oligo(U) tail. The role of the oligo(U) tail and/or editing complex proteins may be especially important for editing the most 5" ESs of an editing block (i.e., the region of editing directed by one gRNA).
Editing activity is focused on the 3' end of the 5' cleavage fragment in both insertion and deletion editing, yet the lack of an upstream base pair dramatically reduces insertion but not deletion editing (11, 20a). This difference may reflect the presentation of insertion versus deletion substrates to the editing catalysts or the use of different catalysis for steps in insertion versus deletion editing (11). The editing complex contains two RNA ligases (24, 27, 32, 33), the significance of which is unclear. However, one is essential for editing (33; A. Schnaufer, unpublished results), and the ligases have been suggested to have differential roles relative to insertion versus deletion editing (32).
Several other proteins of the editing complex have been identified (20, 22, 27a; M. Dro
d, R. Salavati, J. O'Rear, S. S. Palazzo, R. P. Igo, Jr., C. Clayton, and K. Stuart, unpublished data), but only the functions of the RNA ligases and an RNA helicase (26) are evident. However, four related proteins, three of which have zinc finger domains and hence may have RNA and/or protein binding functions, have been identified (27a). This may suggest some functional redundancy in the editing complex. Thus, the enzymatic activities observed here may reflect the composite characteristics of multiple proteins with related activities. Analyses of the catalytic characteristics of the individual enzymes may help to elucidate their roles in editing, although they may be affected by their integration into the editing complex.
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ACKNOWLEDGMENTS |
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This work was supported by NIH Postdoctoral Fellowship AI10312 to R.P.I. and NIH grant GM42188 and HFSPO grant RG/97 to K.S.
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FOOTNOTES |
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REFERENCES |
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