Nucleic Acids Research

Northern blot analysis of RPS12 RNA

The RPS12 gene (formerly called the CR6 gene) is located in the mitochondrial maxicircle DNA and the sequence of fully edited RPS12 RNA predicts a protein with homology to ribosomal protein S12 (27). Trypanosoma brucei RPS12 RNA is edited by the addition of 132 uridines and deletion of 28 uridines (Fig. 1; 7). Exclusive of the poly(A) tract, unedited RPS12 RNA is 221 nt and fully edited RPS12 RNA is 325 nt. Editing creates the start codon, stop codon and open reading frame for this RNA. Partially edited RPS12 RNAs are abundant in the steady-state RNA population (K.Militello and L.Read, unpublished results; this report). It has been previously demonstrated by northern blot experiments that RPS12 RNAs contain both short and long poly(A) tracts (7). Typical RPS12 northern blots are shown in Figure 2A; RPS12 RNA subpopulations with short and long poly(A) tracts are indicated.

A

B

Figure 2. Analysis of RPS12 RNA by northern blotting. (A) Trypanosoma brucei mitochondrial oligo(dT) selected RNA (0.2 µg) was fractionated on a 6% acrylamide-7 M urea gel, transferred to Nytran and probed with a radiolabeled PCR product corresponding to unedited RPS12 sequence (left) or a radiolabeled PCR product corresponding to edited RPS12 sequence (right). RNA subpopulations with short and long poly(A) tracts (7) are indicated. The migration of RNA molecular size standards are displayed on the left. (B) Northern blot analysis of mitochondrial RNAs fractionated on oligo(dT) magnetic beads. Total mitochondrial RNA was fractionated on oligo(dT) beads and equal percentages of oligo(dT) selected RNA [RNA that bound to oligo(dT) beads; ~0.04 µg] and oligo(dT) non-selected RNA [RNA that did not bind to oligo(dT) beads; ~2 µg] were electrophoresed on a 6% acrylamide-7 M urea gel, transferred to Nytran and hybridized to the same probes as used in (A). Lanes marked (+) contained oligo(dT) selected RNAs and lanes marked (-) contained oligo(dT) non-selected RNAs. The migration of RNA molecular size standards are displayed on the left.

The feasibility of using mitochondrial poly(A)⁺ RNA as the source of RPS12 RNA for subsequent experiments was determined by fractionation of total mitochondrial RNA on oligo(dT) beads. RNAs that were captured and subsequently eluted from the oligo(dT) beads were designated oligo(dT) selected RNAs. RNAs that were not captured by the oligo(dT) beads were designated oligo(dT) non-selected RNAs. Equal percentages of both oligo(dT) selected and oligo(dT) non-selected RNAs were analyzed for RPS12 RNA by northern blotting using unedited and edited RPS12 probes (Fig. 2B). The majority (58 ± 18%, n = 4) of RNAs that hybridized to the unedited RPS12 probe were found in the oligo(dT) selected RNA population, demonstrating that the majority of these RNAs contain poly(A) tracts (Fig. 2B). The remaining 42% of the RNA in the oligo(dT) non-selected population may be non-polyadenylated RPS12 RNAs as non-polyadenylated, unedited T.brucei mitochondrial RNAs have previously been reported (5). It is also possible that these RNAs are polyadenylated, but the oligo(dT) capture step is not 100% efficient. Surprisingly, 91 ± 6% (n = 3) of RNAs that hybridized to the edited RPS12 probe were found in the oligo(dT) non-selected RNA population (Fig. 2B). Since it has been demonstrated that these RNAs contain poly(A) tracts by digestion with RNase H and oligo(dT) (7) and it is possible to generate cDNA from these RNAs using oligo(dT) as a primer (Figs 7A and 9), it is likely that these RNAs are not captured efficiently by oligo(dT) beads. Base pairing between the poly(A) tract and uridine-rich regions added by kRNA editing may inhibit binding of the poly(A) tracts to oligo(dT) beads. Since many polyadenylated RPS12 RNAs were not captured efficiently by oligo(dT) beads, we will refer to RNAs as oligo(dT) selected or oligo(dT) non-selected instead of the standard terminology of poly(A)⁺ and poly(A)^-. Based on our northern blot results, the oligo(dT) selected RNAs were the source of RNAs used to analyze the poly(A) tract length of unedited RPS12 RNAs and the oligo(dT) non-selected RNAs were the source of RNAs used to analyze the poly(A) tract lengths of both extensively edited (defined as RNAs edited past site 110; see HinfI restriction site in Fig. 1) and fully edited RPS12 RNAs.

Hybrid selection of RPS12 RNA

An antisense biotinylated oligodeoxynucleotide (CR6-5[prime]biotin) that is complementary to a region in the 5[prime]-portion of the RNA that is never edited (Fig. 1) was used to purify RPS12 RNAs from oligo(dT) selected and oligo(dT) non-selected RNAs as described in Materials and Methods. All RPS12 RNAs have the 5[prime] never edited region, regardless of the extent of editing. RPS12 RNAs purified from oligo(dT) selected RNA were fractionated by gel electrophoresis into four RNA subpopulations, designated subpopulations 1-4 (Fig. 3A). We conclude these RNAs are RPS12 RNAs for the following three reasons. (i) These RNAs are the same sizes as RPS12 RNAs detected by northern blotting (7; compare Figs 3A and 2A). (ii) This pattern was not observed when biotinylated oligodeoxynucleotides antisense to apocytochrome b (28) or NADH dehydrogenase subunit 9 (29) RNAs were used (data not shown). (iii) All four subpopulations of hybrid selected RPS12 RNA could be amplified by RT-PCR using RPS12-specific primers (Figs 4 and 6).

A

B

Figure 3. Purification of RPS12 RNA. RPS12 RNA was purified from either mitochondrial oligo(dT) selected RNA (A) or oligo(dT) non-selected RNA (B) using the oligodeoxynucleotide CR6-5[prime]biotin as described in Materials and Methods. Regions of the gel from which different subpopulations of RNA were eluted are shown on the right. The migration of RNA molecular size standards are displayed on the left. In (B), the film was cut into two pieces due to size constraints of the scanner, accounting for the apparent crack at 140 nt.

RPS12 RNA was also purified from oligo(dT) non-selected RNA (Fig. 3B). These RNAs did not show the characteristic RPS12 pattern observed in RPS12 northern blots (7; Fig. 2A) and the RPS12 RNA hybrid selected from oligo(dT) selected RNAs (Fig. 3A). Instead, heterogeneously sized RNAs were observed which ranged from 100 to 750 nt, although subpopulations of the expected sizes could be faintly discerned. RNAs of three different sizes were purified from the gel. Subpopulation 5 was 300-375 nt which is in the range of fully edited RPS12 RNA with short poly(A) tracts. Subpopulation 6 was 450-600 nt which is in the range of fully edited RPS12 RNA with long poly(A) tracts. RNAs of 100-110 nt were also purified and designated subpopulation X. These RNAs served as a negative control population.

The poly(A) tract sizes of the purified RPS12 RNA sub-populations could not be determined directly using digestion with RNase H and oligo(dT)₁₅ (6) or digestion with RNase T1 (30) due to a lack of material purified from the gel. However, we conclude that subpopulations 1-3 and 5 have short poly(A) tracts since identical size RPS12 RNA populations have short poly(A) tracts as deduced from northern blot analysis of RNase H/oligo(dT) digested RNA (7). By the same criterion, long poly(A) tracts are present on RNAs from subpopulations 4 and 6.

Analysis of purified RPS12 RNAs by RT-PCR and DNA sequencing

The degree of editing of the RPS12 RNAs in the four subpopulations purified from oligo(dT) selected RNA (Fig. 3A) was determined. RNAs from subpopulations 1-4 were amplified by RT-PCR using primers that hybridize to 5[prime] and 3[prime] never edited regions of RPS12 RNA (Fig. 1) and therefore amplify all RPS12 RNAs (Fig. 4). In each case, RT-PCR resulted in products with the sizes expected for RPS12 RNAs (231-335 bp). No products were observed in control reactions without cDNA template. The increasing size of the RT-PCR products from subpopulations 1-3 was consistent with the increasing size of purified RNA (Fig. 3A). RT-PCR products from subpopulation 4 were similar to the sizes of the RT-PCR products from subpopulations 1 and 2.

Figure 4. RT-PCR analysis of RPS12 RNA purified from oligo(dT) selected RNA. RNA subpopulations 1-4 (Fig. 3A) were analyzed for total RPS12 RNA by RT-PCR as described in Materials and Methods. Lane 1, DNA molecular size markers; lane 2, no cDNA control reaction; lane 3, subpopulation 1 cDNA; lane 4, subpopulation 2 cDNA; lane 5, subpopulation 3 cDNA; lane 6, subpopulation 4 cDNA.

The products of each of the four RT-PCR reactions were cloned into the phagemid pBSCII SK- and several randomly chosen clones were analyzed by DNA sequencing (Fig. 5). Clones from subpopulations 1, 2 and 4 were all partially edited. Twelve of 15 clones from subpopulation 3 were partially edited and the remaining three clones were unedited. Despite extensive analysis of the sequence data, we could discern no correlation between the amount of editing and the presence of the long poly(A) tract. In addition, there was no obvious correlation between the presence and/or length of the junction region, defined as incompletely edited sequences at the boundary of unedited and fully edited sequences (31) and any specific RNA subpopulation. However, since partially edited RNAs were found in all four subpopulations, we conclude that partially edited RPS12 RNAs contain both short poly(A) tracts (subpopulations 1-3) and long poly(A) tracts (subpopulation 4).

Figure 5. DNA sequence analysis of RPS12 RNAs purified from oligo(dT) selected RNA. The RT-PCR products from purified subpopulations 1-4 (Fig. 4) were cloned into the phagemid pBSCII SK- and randomly selected clones were analyzed by DNA sequencing. Individual clones are displayed 3[prime]->5[prime] and editing sites are denoted below. Sites 1-7 and 141-165 are not displayed since no editing occurs at these sites in vivo. The first number of each clone is the RPS12 subpopulation from which it originated and the number in parentheses is the experiment number. Asterisks are placed in front of unedited clones. The black bar represents fully edited regions, the light gray bars represent junction regions and the dark gray bars represent unedited regions.

Figure 6. Detection of unedited RPS12 RNA by RT-PCR. RNA subpopulations 1-4 (Fig. 3A) were analyzed for unedited RPS12 RNA by RT-PCR as described in Materials and Methods. The arrow indicates the location of the predicted 239 bp product. Lane 1, DNA molecular size markers; lane 2, no cDNA control reaction (-); lanes 3-6, titration of subpopulation 1 cDNA; lanes 7-10, titration of subpopulation 2 cDNA; lanes 11-14, titration of subpopulation 3 cDNA; lanes 15-18, titration of subpopulation 4 cDNA; lane 19, positive control reaction using a plasmid containing the RPS12 gene as a template (+). The range of cDNA titration for each sample was 1:50, 1:10, 1:5 and 1. The identity of the larger band observed at the highest concentration of subpopulation 2 cDNA is unknown.

A

B

Figure 7. Detection of extensively edited RPS12 RNAs. (A) RT-PCR analysis. RNA subpopulations 5, 6 and X (Fig. 3B) were analyzed for total RPS12 RNA by RT-PCR as described in Materials and Methods. Lane 1, DNA molecular size markers; lane 2, no cDNA control reaction; lanes 3 and 6, subpopulation X cDNA; lanes 4 and 7, subpopulation 5 cDNA; lanes 5 and 8, subpopulation 6 cDNA. cDNA was synthesized using either the CR6-13 primer (lanes 3-5) after treatment with RNase H and oligo(dT)₁₅ or the dT-RXS primer (lanes 6-8). (B) Restriction digestion analysis. kRNA editing creates a HinfI site at editing site 110. The expected sizes of the digestion products of RPS12 RT-PCR products unedited and edited at site 110 are indicated in the cartoon. Subpopulation 5 and 6 RT-PCR products (shown in Fig. 7A) were either left undigested or digested with HinfI. The locations of the 5[prime] and 3[prime] cleavage fragments are indicated by arrows. Lane 1, DNA molecular size markers; lane 2, undigested subpopulation 5 RT-PCR product; lane 3, subpopulation 5 RT-PCR product digested with HinfI; lane 4, undigested subpopulation 6 RT-PCR product; lane 5, subpopulation 6 RT-PCR product digested with HinfI.

To determine the subpopulations that contained the majority of the unedited RPS12 RNA, the four RPS12 RNA subpopulations purified from oligo(dT) selected RNA were analyzed by RT-PCR using unedited RPS12-specific primers with different template concentrations (Fig. 6). The majority of the unedited RPS12 RNAs were found in subpopulation 1 (Fig. 6, lanes 3-6). The expected 239 bp RT-PCR product was generated from this subpopulation even at the lowest cDNA concentration. Unedited RPS12 RNAs were also detected in subpopulations 2-4, but only at the highest cDNA concentration, which is 50-fold higher than the cDNA concentration at which unedited RNAs were detected in subpopulation 1 (Fig. 6, lanes 10, 14 and 18). Moreover, in another experiment, unedited RPS12 RNAs were only detected in subpopulation 1 (data not shown). We therefore conclude that the vast majority of unedited RPS12 RNAs have short poly(A) tracts. Our data indicate that a very small percentage of unedited RPS12 RNAs have long poly(A) tracts. However, we cannot rule out gel artifacts as a cause for this observation and therefore unedited RPS12 RNAs may never have long poly(A) tracts.

RPS12 RNAs purified from oligo(dT) non-selected RNA (Fig. 3B) were analyzed for extensively and fully edited RPS12 RNA. cDNA was synthesized from subpopulations 5, 6 and X using either dT-RXS or CR6-13 oligodeoxynucleotide as a primer. dT-RXS oligodeoxynucleotide is a derivative of oligo(dT) (Materials and Methods) and CR6-13 oligodeoxynucleotide is able to hybridize to the 3[prime]-ends of all RPS12 molecules (Fig. 1). The CR6-13 primer was used in the event the poly(A) tract was blocked by base pairing to uridine-rich regions in the body of the RNA. In this case, the poly(A) tract was first removed by treatment with RNase H and oligo(dT)₁₅ before synthesis of cDNA using the CR6-13 oligodeoxynucleotide as a primer. The purified RPS12 RNAs were amplified by RT-PCR using primers that hybridize to the 5[prime] and 3[prime] never edited regions of RPS12 RNA and therefore amplify all RPS12 RNAs. The results are shown in Figure 7A. No RPS12 RNA was detected in control reactions without cDNA (Fig. 7A, lane 2) or using subpopulation X cDNA (Fig. 7A, lanes 3 and 6) which, based on its migration, would not have been expected to contain RPS12 RNA. Products of the expected size for RPS12 RNA (231-335 bp) were observed in reactions using subpopulation 5 cDNA (Fig. 7A, lanes 4 and 7) and subpopulation 6 cDNA as template (Fig. 7A, lanes 5 and 8). The outcome of the RT-PCR was similar regardless of whether the dT-RXS or CR6-13 oligodeoxynucleotide primed cDNA was used as a template. Although these subpopulations originate from oligo(dT) non-selected RNA, they do contain poly(A) tracts as demonstrated by the ability of oligo(dT) to prime cDNA synthesis for RT-PCR analysis.

To determine if extensively edited RPS12 RNAs were present in subpopulations 5 and 6, the RT-PCR products were digested with the restriction enzyme HinfI. kRNA editing creates a unique HinfI site at editing site 110 (Fig. 1). If the RNA is not edited at site 110, the RT-PCR product will not be digested (Fig. 7B). If the RNA is edited at site 110, the RT-PCR product will be digested into a 219 bp 3[prime] fragment and a variable length 5[prime] fragment, ranging from 78-115 bp depending on the extent of editing 5[prime] to site 110. A small percentage of the subpopulation 5 RT-PCR product is digested by HinfI indicating that some of the RPS12 RNAs in this subpopulation are edited at site 110 (Fig. 7B, lanes 2 and 3). It is unlikely that the restriction digestion was incomplete as these conditions allow almost complete digestion of subpopulation 6 RT-PCR products (Fig. 7B, lanes 4 and 5). Restriction digestion of subpopulation 5 RT-PCR product using a restriction enzyme that recognizes all RPS12 DNAs demonstrated that the RT-PCR product is indeed RPS12 DNA (data not shown). HinfI almost completely digested subpopulation 6 RT-PCR product, indicating that the majority of the RPS12 RNAs in this subpopulation are edited at site 110 (Fig. 7B, lanes 4 and 5). We conclude that extensively edited RPS12 RNAs have both short poly(A) tracts (subpopulation 5) and long poly(A) tracts (subpopulation 6).

The sequence composition of the RPS12 RNAs in the two subpopulations purified from oligo(dT) non-selected RNA was determined. The RT-PCR products (Fig. 7A) were cloned into the phagemid pBSCII SK- and several randomly chosen clones were analyzed by DNA sequencing (Fig. 8). Eleven of 12 subpopulation 5 clones were partially edited. The remaining clone was unedited. Eight of eight subpopulation 6 clones were partially edited. The size of sequenced clones from subpopulation 5 reflected the size of the subpopulation 5 RT-PCR products. However, although the majority of the RT-PCR products from subpopulation 6 appeared to be edited at site 110 by digestion with HinfI (Fig. 7B), sequence analysis of these RT-PCR products indicated that only one of eight clones [6p-4(1)] was edited to site 110. Therefore, a bias may exist that promotes cloning of less edited RT-PCR products in this sample. Again, there was no correlation between the presence and/or length of the junction region and a specific RNA subpopulation. All RNAs in subpopulation 6 were edited to some extent, indicating that the presence of editing is correlated with the presence of the long poly(A) tract. This observation is consistent with the sequence analysis of the subpopulation 4 RNAs (Fig. 5). However, it is difficult to make conclusions regarding any correlation between the amount of editing and the presence of the long poly(A) tract due to the cloning bias. Nonetheless, these data provide further evidence that partially edited RNAs contain both short poly(A) tracts (subpopulation 5) and long poly(A) tracts (subpopulation 6).

Figure 8. DNA sequence analysis of RPS12 RNAs purified from oligo(dT) non-selected RNA. The subpopulation 5 and 6 (Fig. 7A) RT-PCR products were cloned into the phagemid pBSCII SK- and randomly selected clones were chosen for DNA sequencing. Individual clones are displayed 3[prime]->5[prime] and the positions of editing sites are denoted below. Sites 1-7 and 141-165 are not displayed since no editing occurs at these sites in vivo. The first number of each clone is the RPS12 subpopulation from which it originated and the number in parentheses is the experiment number. Asterisks are placed in front of unedited clones. The black bar represents fully edited regions, the light gray bars represent junction regions and the dark gray bars represent unedited regions.

RT-PCR was used to determine the location of the fully edited RPS12 RNAs in the two subpopulations purified from oligo(dT) non-selected RNA (Fig. 9). Primers specific to fully edited RPS12 RNA were used with varying concentrations of cDNA template. No fully edited RT-PCR product was generated using subpopulation X cDNA (data not shown) or in a reaction containing no cDNA template (Fig. 9, lane 2). A 302 bp fully edited RT-PCR product was generated using both subpopulation 5 cDNA (Fig. 9, lanes 3-6 and 11-14) and subpopulation 6 cDNA (Fig. 9, lanes 7-10 and 15-18). Again, the outcome of the RT-PCR was similar regardless of whether dT-RXS or CR6-13 oligodeoxynucleotide primed cDNA was used. We conclude that fully edited RPS12 RNAs contain both short poly(A) tracts (subpopulation 5) and long poly(A) tracts (subpopulation 6). The ratio of fully edited RPS12 RNAs containing short and long poly(A) tracts varied between experiments (data not shown).

DISCUSSION

Trypanosoma brucei mitochondrial RNAs are present in populations containing both short and long poly(A) tracts and poly(A) tract lengths are often developmentally regulated (6-9). However, because of the complexity added to the mitochondrial RNA population by kRNA editing, previous northern blot experiments have not been able to distinguish precisely which sequence classes of a given RNA (unedited, partially edited or fully edited) contain which poly(A) tract lengths. RPS12 RNAs were purified by hybrid selection and separated by gel electrophoresis into populations that differ in poly(A) tract length. RT-PCR and DNA sequencing analysis of the hybrid selected RNAs allowed us to determine the sequence classes of RPS12 RNAs present in these populations and therefore to determine the relationship between the presence of kRNA editing and poly(A) tract length. Unedited RPS12 RNAs were found almost exclusively in subpopulations that contain short poly(A) tracts (Fig. 6). These results are consistent with northern blot experiments demonstrating that RNAs hybridizing to unedited probes contain short poly(A) tracts (7). Partially edited and fully edited RPS12 RNAs were found in populations that contain both short and long poly(A) tracts (Figs 5, 8 and 9). These results are consistent with northern blot experiments in which probes complementary to edited RNA sequences detected RNAs with both poly(A) tract lengths (6-9).

Figure 9. Detection of fully edited RPS12 RNA by RT-PCR. RNA subpopulations 5 and 6 were analyzed for fully edited RPS12 RNA by RT-PCR as described in Materials and Methods. The arrow indicates the location of the predicted 302 bp product. Lane 1, DNA molecular size markers; lane 2, no cDNA control reaction (-); lanes 3-6 and 11-14, titration of subpopulation 5 cDNA; lanes 7-10 and 15-18, titration of subpopulation 6 cDNA; lane 19, positive control reaction using a plasmid containing the fully edited RPS12 cDNA as a template (+). The range of cDNA titration for each sample was 1:100, 1:50, 1:10 and 1. cDNA was synthesized using either the CR6-13 oligodeoxynucleotide after treatment with RNase H and oligo(dT)₁₅ (lanes 3-10) or the dT-RXS oligodeoxynucleotide (lanes 11-18).

In many systems, an increase in the length of the poly(A) tract is correlated with an increase in the translation of a specific RNA (for a review see 10,13-16). We originally speculated that the long poly(A) tract might be a signal for translation in the mitochondrion of T.brucei and could provide a mechanism by which only translatable RNAs associate with ribosomes. In this system, only never edited and fully edited RNAs are predicted to be translated since they are the only RNAs with complete open reading frames. Thus, this hypothesis predicts that only fully and never edited RNAs would possess long poly(A) tracts. However, the present study indicates that some partially edited RPS12 RNAs contain long poly(A) tracts. Therefore, the long poly(A) tract is unlikely to provide a mechanism by which only mature RNAs are translated in T.brucei mitochondria. It cannot be ruled out, however, that long poly(A) tracts are required for translation, but that an additional signal present only in fully and never edited RNAs is also required. The establishment of an in vitro T.brucei mitochondrial translation system will be necessary to test these hypotheses.

As an alternative to, or in addition to, a role in translation, polyadenylation may regulate RNA stability in the mitochondrion of T.brucei. The two poly(A) tract sizes could provide differential stability to different subpopulations of mitochondrial RNAs. One mechanism by which this may be accomplished is suggested by northern blot data of oligo(dT) selected and oligo(dT) non-selected mitochondrial RNA (Fig. 2A). A significant proportion of extensively and fully edited RPS12 RNAs were unable to bind to oligo(dT) beads even though RT-PCR experiments demonstrated that these RNAs contained poly(A) tracts. We speculate that base pairing occurs between the poly(A) tract and uridine-rich regions added by kRNA editing and that this base pairing blocks association of the poly(A) tract with oligo(dT) beads. If this RNA structure exists in vivo, it could regulate the stability of uridine-rich RNAs. Double-stranded RNA structures are likely to be more resistant to nuclease digestion. The poly(A) tract could also regulate RNA stability through interactions with specific proteins such as poly(A)-binding proteins (for a review see 11) or poly(A) nucleases (32). Pulse-chase RNA stability experiments will be necessary to analyze the role of the poly(A) tract length in the stability of RNAs in T.brucei mitochondria and these experiments are in progress in our laboratory. Polyadenylation may also play a role in processes other than translation and RNA stability. However, it is unlikely that polyadenylation regulates kRNA editing efficiency, since most never edited RNAs also contain both short and long poly(A) tracts (6).

The presence of kRNA editing is correlated with the presence of long poly(A) tracts since unedited RPS12 RNAs were found almost exclusively in subpopulations that contain short poly(A) tracts but partially edited and fully edited RPS12 RNAs were found in subpopulations containing both short and long poly(A) tracts. There are several mechanisms by which these two processes could be functionally connected. kRNA editing could create a cis-acting regulatory sequence in the RNA that is required for long poly(A) tract synthesis. Alternatively, the kRNA editing machinery (termed the editosome), which presumably assembles on the 3[prime]-end of the RNA, could physically block the machinery that adds the long poly(A) tracts. As editing proceeds 3[prime]->5[prime], the 3[prime]-end of the RNA would be freed and the long poly(A) tract added. The editosome could also recruit the poly(A) polymerase and associated factors to the unedited RNA. All of these models suggest that the kRNA editing machinery and the polyadenylation machinery are physically associated or are able to process a given RNA concurrently.

The results presented here demonstrate that a portion of partially edited RPS12 RNAs contain long poly(A) tracts. Thus, in T.brucei mitochondria, the long poly(A) tract is probably not the lone signal for translation. The correlation between the presence of kRNA editing and the presence of a long poly(A) tract suggests that multiple RNA processing events in the mitochondria of T.brucei are coordinated. A thorough examination of the mechanisms by which these RNA processing events interact will increase our understanding of the unique methods of gene regulation in trypanosome mitochondria.

ACKNOWLEDGEMENTS

We thank Paul Gollnick, Margaret Hollingsworth, Mark Hayman and Michel Pelletier for critical reading and discussion of the manuscript. This work was supported in part by NIH grant GM53502 to L.K.R. who is also a recipient of the Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology.

REFERENCES

1. Stuart,K. and Feagin,J.E. (1992) Int. Rev. Cytol., 141, 65-88.

2. Stuart,K., Allen,T.E., Heidmann,S. and Seiwert,S.D. (1997) Microbiol. Mol. Biol. Rev., 61, 105-120.

3. Abraham,J.M., Feagin,J.E. and Stuart,K. (1988) Cell, 55, 267-272. MEDLINE Abstract

4. Sturm,N.R. and Simpson,L. (1990) Cell, 61, 871-878. MEDLINE Abstract

5. Decker,C.J. and Sollner-Webb,B. (1990) Cell, 61, 1001-1011. MEDLINE Abstract

6. Bhat,G.J., Souza,A.E., Feagin,J.E. and Stuart K. (1992) Mol. Biochem. Parasitol., 52, 231-240. MEDLINE Abstract

7. Read,L.K., Myler,P.J. and Stuart,K. (1992) J. Biol. Chem., 267, 1123-1128. MEDLINE Abstract

8. Read,L.K., Stankey,K.A., Fish,W.R., Muthiani,A.M. and Stuart,K. (1994) Mol. Biochem. Parasitol., 68, 297-306.

9. Bhat,G.J., Myler,P.J. and Stuart,K. (1991) Mol. Biochem. Parasitol., 48, 139-150. MEDLINE Abstract

10. Monroe,D. and Jacobson,A. (1990) Gene, 91, 151-158. MEDLINE Abstract

11. Jackson,R.J. and Standart,N. (1990) Cell, 62, 15-24. MEDLINE Abstract

12. Sachs,A. and Wahle,E. (1993) J. Biol. Chem., 268, 22955-22958. MEDLINE Abstract

13. Deshpande,A.K., Chatterjee,B. and Roy,A.K. (1979) J. Biol. Chem., 254, 8937-8942. MEDLINE Abstract

14. Monroe,D. and Jacobson,A. (1990) Mol. Cell. Biol., 10, 3441-3455. MEDLINE Abstract

15. Sheets,M.D., Fox,C.A., Hunt,T., Vande Woude,G. and Wickens,M. (1994) Genes Dev., 8, 926-938. MEDLINE Abstract

16. Sallës,F.J., Lieberfarb,M.E., Wreden,C., Gergen,J.P. and Strickland,S. (1994) Science, 266, 1996-1999. MEDLINE Abstract

17. Cohen,S. (1995) Cell, 80, 829-832. MEDLINE Abstract

18. Sarker,N. (1997) Annu. Rev. Biochem., 66, 173-197. MEDLINE Abstract

19. Kudla,J., Hayes,R. and Gruissem,W. (1996) EMBO J., 15, 7137-7146. MEDLINE Abstract

20. Lisitsky,I., Klaff,P. and Schuster,G. (1996) Proc. Natl Acad. Sci. USA, 93, 13398-13403. MEDLINE Abstract

21. Lisitsky,I., Kotler,A. and Schuster,G. (1997) J. Biol. Chem., 272, 17648-17653. MEDLINE Abstract

22. Stuart,K., Gobright,E., Jenni,L., Milhausen,M., Thomashow,L. and Agabian,N. (1984) J. Parasitol., 70, 747-754. MEDLINE Abstract

23. Brun,R. and Schönenberger,M. (1979) Acta Trop., 36, 289-292.

24. Harris,M.E., Moore,D.R. and Hajduk,S.L. (1990) J. Biol. Chem., 265, 11368-11376. MEDLINE Abstract

25. Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156-159. MEDLINE Abstract

26. Min,J. and Zassenhaus,P.Z. (1992) BioTechniques, 13, 870-874. MEDLINE Abstract

27. Maslov,D.A., Sturm,N.R., Niner,B.M., Gruszynski,E.S., Peris,M. and Simpson L. (1992) Mol. Cell. Biol., 12, 56-67. MEDLINE Abstract

28. Feagin,J.E., Shaw,J.M., Simpson,L. and Stuart,K. (1988) Proc. Natl Acad. Sci. USA, 85, 539-543. MEDLINE Abstract

29. Souza,A.E., Shu,H.-H., Read,L.K., Myler,P.J. and Stuart,K.D. (1993) Mol. Cell. Biol., 13, 6832-6840.

30. Ingle,C.A. and Kushner,S.R. (1996) Proc. Natl Acad. Sci. USA, 93, 12926-12931. MEDLINE Abstract

31. Koslowsky,D.J., Bhat,G.J., Read,L.K. and Stuart,K. (1991) Cell, 67, 1-20. MEDLINE Abstract

32. Sachs,A. and Deardorff,J.A. (1992) Cell, 70, 961-973. MEDLINE Abstract

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*To whom correspondence should be addressed. Tel: +1 716 829 3307; Fax: +1 716 829 2158; Email: lread@acsu.buffalo.edu

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