©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Accurate Modification of the Trypanosome Spliced Leader Cap Structure in a Homologous Cell-free System (*)

(Received for publication, May 9, 1995)

Elisabetta Ullu (1) (2) Christian Tschudi (1)(§)

From the  (1)Department of Internal Medicine and (2)Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8022

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

During RNA maturation in trypanosomatid protozoa, trans-splicing transfers the spliced leader (SL) sequence and its cap from the SL RNA to the 5` end of all mRNAs. In Trypanosoma brucei and Crithidia fasciculata the SL RNA has an unusual cap structure with four methylated nucleotides following the 7-methylguanosine residue (cap 4). Since modification of the 5` end of the SL RNA is a pre-requisite for trans-splicing activity in T. brucei, we have begun to characterize the enzyme(s) involved in this process. Here we report the development of a T. brucei cell-free system for modification of the cap of the SL RNA. Analysis of the nucleotide composition of the in vitro generated cap structure by two-dimensional thin layer chromatography established that the in vitro reaction is accurate. Cap 4 formation requires the SL RNA to be in a ribonucleoprotein particle and can be inhibited by annealing a complementary 2`-O-methyl RNA oligonucleotide to nucleotides 7-18 of the SL RNA. Methylation of the 5` end of the SL RNA is also required for trans-splicing in T. cruzi and Leishmania amazonensis and cell-free extracts from C. fasciculata and L. amazonensis are capable of modifying the cap structure on the T. brucei SL ribonucleoprotein particle.


INTRODUCTION

In eukaryotes virtually all RNA molecules, whether transcribed by RNA polymerase I, II, or III, undergo post-transcriptional modifications. With the exception of tRNA molecules, which contain a high number and a complex variety of modified nucleosides, all other RNAs analyzed to date are modified by addition of methyl groups either to the ribose moiety, to the base, or to both, or by modification of uridine to pseudouridine. The significance of these modified nucleotides in the function and metabolism of RNAs other than tRNAs is not fully understood. However, there are a few cases that indicate an important functional role. These include, but are not limited to, the following examples: the 7-methylguanosine (m^7G) (^1)cap of mRNA is required for assembly of the translation initiation complex(1) ; trimethylation of the guanosine cap structure of vertebrate small nuclear RNAs appears to function as part of a nuclear location signal of newly assembled small nuclear ribonucleoprotein particles(2, 3) ; methylation of the 2` position of the ribose at a conserved guanosine residue in the large mitochondrial ribosomal RNA of Saccharomyces cerevisiae is required for assembly of a functional 50 S subunit (4) ; in Escherichia coli discrimination between tRNA and tRNA is mediated by a modification specific to the initiator tRNA(5) . In addition, there is circumstantial evidence for a functional role of modified ribonucleotides. For instance, spliceosomal small nuclear RNAs (snRNAs) are highly modified and the modifications are clustered in areas of the molecule which have been shown by genetic and biochemical means to be essential for function(6) . Examples include the very 5` end of U1 snRNA, the 5`-half of U2 snRNA, the conserved loop of U5 snRNA, and the conserved ACAGAG sequence of U6 snRNA.

In Trypanosoma brucei, the biogenesis of a functional spliced leader (SL) RNA includes, as an essential pre-requisite, extensive modifications of the SL RNA 5` end. Unlike any other eukaryotic cap structure which has no more than two modified nucleotides, the SL cap has four consecutive modified nucleotides. By convention this highly unusual 5` terminus is referred to as a cap 4 structure(7, 8, 9, 10) . The only other modified position of the SL RNA is the adenosine residue at position 6 which carries a methyl group at the 2` position of the ribose(8) . Detailed structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry determined the SL cap 4 structure to be m^7guanosine(5`)ppp(5`)-N^6,N^6,2`-O-trimethyladenosine-p-2`O-methyladenosine-p-2`-O-methylcytosine-p-3,2`-O-dimethyluridine (7) . The N^6,N^6,2`-O-trimethyladenosine and the 3,2`-O-dimethyluridine modifications represent nucleotides previously unknown in nature. Although no clear functional role has yet been demonstrated for the SL cap 4 structure, its importance is suggested by several observations. First, trans-splicing transfers the first 39 nucleotides (nt) of the SL RNA, including the cap 4 structure, to the 5` end of every mRNA and thereby stabilizes the mRNA against degradation (11, 12, 13) . Second, using permeable cells we have shown that modification of the SL cap is essential for utilization of the SL RNA in trans-splicing(14) . This result was further supported by preventing modification of the cap 4 structure by a different approach, namely by binding an antisense oligonucleotide to nt 7-18 of the SL sequence(15) , and by in vivo studies with the methylation inhibitor sinefungin(16) . Third, by analogy to the function of the mRNA cap in other eukaryotic organisms, we would expect the m^7G cap of the SL RNA to be required for the assembly of the translation initiation complex. Thus, the mechanism of cap 4 formation and its function in mRNA metabolism has become a focal point of our research.

This study describes the establishment of a T. brucei cell-free system for the accurate modification of the SL RNA 5` end. The experiments presented here show that cap formation requires the SL RNA to be in a ribonucleoprotein particle (RNP) and that trypanosomatid protozoa share a common machinery for the modification of this cap structure.


MATERIALS AND METHODS

RNA Synthesis and Analysis

Cultured procyclic trypanosome cells were grown and permeabilized essentially as described(14, 17) . For cap analysis, P-labeled RNA was synthesized under standard conditions(14) , except that all four ribonucleotide triphosphates were included at a concentration of 1 mCi/ml (3000 Ci/mmol; Amersham) and no unlabeled triphosphates were included in the transcription reaction.

For RNP preparations, cells were pelleted at the end of RNA synthesis, resuspended in transcription buffer containing 0.5 mMS-adenosyl-L-homocysteine (Ado-Hcy) and 2 mM ATP, and lysed by the addition of Nonidet P-40 to 0.5%. RNPs were enriched by chromatography on a DEAE-Sepharose CL-6B column as described(14) .

Permeabilization and RNA synthesis in Trypanosoma cruzi epimastigotes and Leishmania amazonensis promastigotes was done by adapting our established protocol for T. brucei cells(17) . RNase mapping probes for the respective SL RNAs were generated by the polymerase chain reaction and are complementary to nt 7 to 105 of the T. cruzi SL RNA and to nt 7 to 90 of the L. amazonensis SL RNA.

In Vitro Assay for Cap 4 Synthesis

Two different methods were used to prepare cell-free extracts from procyclic T. brucei cells. In the first procedure, we essentially followed the procedure of Dignam et al. (18) and prepared crude nuclear and cytoplasmic extracts. Alternatively, we used mild sonication of T. brucei cells in transcription buffer (17) to generate whole cell extracts. Both procedures gave extracts active in modification. Crithidia and Leishmania extracts were prepared by the procedure of Dignam et al. (18) and by mild sonication, respectively. The modification reaction was done in 20 µl, using 5-13 µl of extract (1-10 mg of protein/ml), along with 20 mM HEPES-KOH (pH 7.8), 3 mM MgCl(2), 150 mM sucrose, 1 mM dithiothreitol, 10 µg/ml leupeptin, 0.5 mMS-adenosyl-L-methionine, and P-labeled SL RNP. After incubation for 30 min at 30 °C, RNA was first extracted with a solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% N-lauroylsarcosine, then with water-saturated phenol, precipitated with ethanol, and either analyzed by RNase protection (14) or digested with a mixture of RNases as described below.

Analysis of Cap 4 Structure

Radiolabeled RNA was digested in 50 mM NH(4)OAc (pH 4.5) and 2 mM EDTA with 20 µg/ml T1, 20 units/ml T2, and 200 µg/ml RNase A at 37 °C for 14 h (50 µl total volume). The digestion products were then dried under vacuum and fractionated directly on a 25% polyacrylamide, 7 M urea gel. For a structural analysis of the SL cap 4, the products of the mixed RNase digestion were applied to a DEAE-Sepharose CL-6B column (0.2-ml bed volume in 10 mM ammonium formate, pH 8). After washing with 10 mM ammonium formate, the column was step eluted with 3 column volumes each of 0.2, 0.3, 0.35, 0.4, and 0.5 M ammonium formate. Under these conditions, the T2-resistant cap 4 structure elutes at 0.5 M ammonium formate, essentially free of radiocontaminants. Fractions containing purified cap 4 were lyophilized, resuspended in water, and dried once more. The samples were finally resuspended in 40 µl of H(2)O, heated to 100 °C for 3 min, and chilled on ice. 30 µg of yeast tRNA was added and the samples were digested for 3 h at 45 °C with nuclease P1 (Sigma; 5 units) in 10 mM NH(4)OAc, pH 5.3 (50 µl total volume). The pH was adjusted to 7.5 by the addition of 5 µl of 1 M NH(4)HCO(3) and after addition of nucleotide pyrophosphatase (Sigma; 2 milliunits) incubation was continued for another 2 h at 37 °C. The digestion products were then chromatographed on cellulose thin layer plates using as a solvent isobutyric acid/concentrated NH(4)OH/H(2)O, 66:1:33 (v/v/v), in the first dimension, and 0.1 M sodium phosphate (pH 6.8), ammonium sulfate, 1-propanol, 100:60:2 (v/w/v), for the second dimension.


RESULTS

Assays for Modifications

We have previously shown that methylation of the SL cap 4 structure is required for utilization of the SL RNA in trans-splicing by blocking de novo methylations in permeable trypanosome cells with Ado-Hcy(14) . To monitor the extent of modification of newly synthesized and radiolabeled SL RNA, we had established a ribonuclease (RNase) protection assay with RNase A and T1 using an antisense SL RNA probe that extends from nt 7 to 128 of T. brucei SL RNA(14) . This probe does not include the first six nucleotides complementary to the sequence AACUAA, where the modified nucleotides are located. The two pyrimidine residues are cleavage sites for RNase A. But if these two nucleotides carry 2`-O modifications, RNase A will not cleave and we expect a protected fragment of 129 nt (referred to as modified SL RNA). In contrast, a shorter protected fragment of 124 nt (referred to as unmodified SL RNA) is diagnostic of SL RNA without the above modifications. However, since this assay only detects 2`-O modifications at positions 3 and 4 of the SL RNA, we also used a procedure originally described by Bangs et al. (7) to monitor 2`-O modifications at positions 1-4. Ribonuclease T2 cleaves RNA at every position, but does not hydrolyze pyrophosphate bonds or 5` bonds adjacent to 2`-O-modified nucleosides. Consequently, digestion of T. brucei SL RNA with T2 generates a T2-resistant fragment with the sequence m^7GpppAACUAp, since m^7G is followed by four 2`-O-modified nucleosides and the fifth residue (A) is not modified(7) . This T2-resistant fragment can then be easily separated from 3`-phosphate mononucleotides by electrophoresis through a 7 M urea, 25% polyacrylamide gel (Fig. 2A). Indeed, if P-labeled SL RNA synthesized in permeable trypanosome cells is gel purified and digested to completion with T2, the expected T2-resistant fragment is obtained (data not shown). More importantly, the SL cap is the largest T2-resistant structure generated when P-labeled total RNA of T. brucei is digested with mixed RNases and fractionated on a 25% denaturing polyacrylamide gel (Fig. 2A, lane 3).


Figure 2: T2 RNase digestion of capped RNA. A, the SL 5` end was modified in a T. brucei nuclear extract and after digestion with T2, the products were separated on a 25% polyacrylamide, 7 M urea gel. The T2-resistant structure (T2R) of in vitro modified SL RNA (lane 2) and of SL RNA synthesized in permeable cells (lane 3) is indicated. An RNase T2 digestion of unmodified RNA that was not incubated in extracts is provided for comparison (lane 1). In lanes 2 and 3, a T2-resistant fragment of faster mobility is also visible. Its identity is at present unclear. B, DEAE-Sepharose chromatography of the T2-resistant cap structure. In vitro modified SL RNA was digested to completion with T2 and fractionated on DEAE-Sepharose CL-6B as described(7) . An aliquot of the onput fraction is shown in lane ON. The column was step-eluted with the concentrations of ammonium formate indicated above the figure. Nucleoside 3`-monophosphates elute predominantly at 0.2 M ammonium formate and only an aliquot of one such fraction is shown.



Establishment of a T. brucei Cell-free System

In attempting to develop a cell-free system for SL-specific cap modifications, we reasoned that for the assay we would initially use the ionic conditions we established for transcription and RNA processing in permeable trypanosome cells(14, 17) . Under these conditions, namely low potassium and low magnesium ions, the SL RNA is efficiently synthesized, modified, assembled in a ribonucleoprotein particle, and utilized for trans-splicing(14, 17) . As a substrate for modification we decided to employ P-labeled SL RNP, synthesized in permeable T. brucei cells in the presence of 0.5 mM Ado-Hcy to prevent modification of the SL RNA 5` end(14) . This decision was taken on the basis of several considerations. First, we and others have been unable to synthesize in vitro, with the aid of phage RNA polymerases, SL RNA with the proper 5` end, since the first nucleotide of the SL RNA is an A residue and not a G residue, which is the preferred initiating nucleotide by phage polymerases. Second, Cross et al.(19) have reported, and we have confirmed this finding, (^2)that if proteins are removed from the SL RNA, the first 20 nt of the SL sequence are no longer digested by RNase H when bound to a complementary DNA oligonucleotide. This suggested the possibility that protein(s) might affect the secondary and perhaps the tertiary structure of the SL RNA. Third, we reasoned it was likely that the SL RNP was the in vivo substrate for the modifications. A fraction enriched for ``undermodified'' SL RNP was prepared by DEAE-Sepharose chromatography as described(14) . This RNP preparation is not completely devoid of modifications since approximately 50% of the SL RNA molecules carry a m^7G cap, whereas the remainder is capped with an unmodified G residue(14) . However, the four nucleotides after the cap are not detectably modified. Using RNase mapping (Fig. 1, lane 1) and T2 digestion (Fig. 2A, lane 1), we confirmed that the first four nucleotides of the SL RNP substrate do not contain any detectable modification. Incubation of this RNP preparation in two different whole cell-free extracts from T. brucei, followed by RNase mapping, resulted in protected fragments that were diagnostic of 2`-O modifications at positions 3 and 4 of the SL RNA (Fig. 1, lanes 3 and 4). A similar result was obtained with a 10,000 g supernatant from extract 2 (lane 5). Inclusion of 0.5 mM Ado-Hcy completely abolished the appearance of these fragments (Fig. 1, lane 2), indicating that methyl groups were being added to the SL RNA. To further characterize the in vitro modified SL RNA, RNase-protected SL RNA fragments corresponding to modified SL RNA were prepared and digested with ribonuclease T2, and the products of digestion fractionated by denaturing gel electrophoresis. This analysis showed that the SL RNP modified in the extract gives rise to a predominant T2-resistant fragment which co-migrates with the one originating from total RNA synthesized in permeable cells (data not shown).


Figure 1: In vitro modification of the SL RNA cap structure. RNase protection analysis of radiolabeled SL RNP and SL RNA after incubation in T. brucei sonic extracts. Unmodified P-labeled SL RNP was incubated without extract (lane 1), in whole cell extract 2 in the presence of Ado-Hcy (lane 2), in whole cell extract 1 (lane 3), in whole extract 2 (lane 4), and in the 10,000 g supernatant of extract 2 (lane 5). P-Labeled SL RNA isolated from the RNP fraction was incubated in extract 1 (lane 6) or extract 2 (lane 7). Lane C shows the modified and unmodified fragments of P-labeled SL RNA from permeable trypanosome cells as described previously(14) . The SL RNA fragments were separated on a 6% polyacrylamide, 7 M urea gel and detected by autoradiography.



To facilitate the detection of the T2-resistant fragment, we omitted RNase mapping in subsequent experiments. An example of such an assay, where total RNA and in vitro modified RNA were digested with T2, is shown in Fig. 2A. Since we have previously noted that the absence of methyl groups increases the electrophoretic mobility of the SL RNA under denaturing conditions (14) , (^3)the comigration of control and in vitro synthesized T2-resistant fragments (compare lanes 2 and 3 in Fig. 2A) suggested that the in vitro modification reaction is accurate, at least in terms of the total number of methyl groups added.

Next, we determined the precise nucleotide composition of the T2-resistant fragment by two-dimensional thin layer chromatography TLC (Fig. 3). For this experiment RNA synthesis in permeable cells was carried out in the presence of all four P-labeled nucleotide triphosphates. T2-resistant fragments from control RNA and from the RNP substrate after in vitro modification were isolated by chromatography on DEAE-Sepharose columns (Fig. 2B) and an equal number of counts were further digested to mononucleotides with nucleotide pyrophosphatase and nuclease P1. As predicted from its known structure (m^7guanosine(5`)ppp(5`)-N^6,N^6,2`-O-trimethyladenosine-p-2`-O-methyladenosine-p-2`-O-methylcytosine-p-3,2`-O-dimethyluridine-p-adenosine-p), the T2-resistant fragment of control RNA gave rise to six spots, four of which comigrated with markers for m^7G (pm^7G), adenosine 5`-monophosphate (pA), 2`-O-methyladenosine 5`-monophosphate (pAm), and 2`-O-methylcytosine 5`-monophosphate (pCm). The remaining two spots represent modified adenosine and uridine residues, as determined by TLC analysis of T2-resistant fragments labeled either with [alpha-P]ATP or [alpha-P]UTP (data not shown). Since the relative mobilities of these two spots are indistinguishable from N^6,N^6,2`-O-trimethyladenosine 5`-monophosphate and 3,2`-O-dimethyluridine 5`-monophosphate previously published for in vivo labeled SL RNA using the same TLC system(8) , we concluded that the SL T2-resistant fragment synthesized in permeable cells carries the proper modified nucleotides. More importantly, the T2-resistant structure generated in the in vitro extract gives rise to six nucleotides with mobilities identical to those derived from control RNA (compare panel A and B in Fig. 3). Taken together, these results demonstrated that the in vitro reaction accurately mimics the SL-specific modifications taking place in vivo.


Figure 3: Analysis of cap 4 constituents by two-dimensional TLC. T2-resistant fragments from control SL RNA synthesized in permeable cells (A) or from substrate SL RNA after modification in a T. brucei nuclear extract (B) were purified over a DEAE-Sepharose column as shown in Fig. 2B and the 0.5 M eluate was digested with nucleotide pyrophosphatase and nuclease P1. The digestion products were separated on cellulose TLC as described(8) . The positions of some representative nonradioactive markers (visualized by UV irradiation) are indicated. The different intensities of the spots are due to differences in the pools of endogenous ribonucleotide triphosphates.



Parameters of the in Vitro Reaction

Our initial incubation mixture closely resembled the transcription buffer we use for RNA synthesis in permeable cells(14) , which includes among other ingredients, creatine phosphate (25 mM), creatine kinase (0.6 mg/ml), 3 mM MgCl(2), 2 mM ATP, 1 mM each of GTP, CTP, and UTP, 20 mM KCl, and 0.5 mMS-adenosyl-L-methionine. We have tested several of these components and found that creatine phosphate, creatine kinase, and the four ribotriphosphates are not required when using a crude cell extract (data not shown). In terms of extract preparation, we have successfully used whole cell, nuclear, or cytoplasmic extracts. Finally, the SL-specific modifications are strongly inhibited by KCl concentrations above 150 mM (Fig. 4).


Figure 4: Salt sensitivity of the SL-specific methyltransferases. In vitro modification in a T. brucei nuclear extract was carried out at various KCl concentrations as indicated above each lane and the SL T2-resistant fragment (T2R) was analyzed by electrophoresis through a 25% denaturing polyacrylamide gel. no extr. lane, input RNA.



To test whether the SL RNA without its associated proteins could be used as a substrate for in vitro modification, we deproteinized the RNP preparation used for the assay and incubated the resulting RNA in the extract. According to the RNase protection test, deproteinized SL RNA was not a substrate for modification (Fig. 1, lanes 6 and 7). This was not due to an inhibitory substance present in the deproteinized RNA, since addition of the purified RNA to the RNP preparation did not inhibit the appearance of modified SL RNA (data not shown).

Synthesis of modified SL RNA was dependent upon addition of the extract since incubation of the RNP fraction in buffer alone does not result in modification of the SL RNA 5` end (Fig. 1, lane 1). Lastly, in agreement with our previous observations (15) annealing of a complementary 2`-O-methyl RNA oligonucleotide to nt 7-18 of the SL RNP blocks the appearance of the SL+T2-resistant structure (Fig. 5).


Figure 5: Cap modification is inhibited by occlusion of sequences between nt 7 and 18 of the SL RNA. In vitro modifications were carried out in the presence of 2`-O-methyl RNA oligomers complementary to nt 7-18 (RZ, lane 3) and to nt 110-119 (RS, lane 4) of the SL RNA(15) . A control reaction without added oligos is shown in lane 2.



A Similar Mechanism of SL Cap Modification in Trypanosomatid Protozoa

The conservation of the cap 4 between such divergent genera as T. brucei and Crithidia fasciculata(7) raises the possibility that its structure and function are common to all trypanosomatid protozoa. To begin to address this issue, we tested whether methylation of the SL RNA plays a role in T. cruzi and L. amazonensis trans-splicing. We developed permeable cell systems for these two organisms and monitored the extent of modification of newly synthesized SL RNA by RNase mapping of total P-labeled RNA as described above for T. brucei. In both cases, the antisense SL RNA probe did not include the first six nucleotides complementary to the sequence AACUAA, which are the sites of modifications in T. brucei. Under the conditions used, T. cruzi and L. amazonensis cells expressed abundant SL RNA, and RNase mapping of this RNA revealed protected fragments corresponding to both modified and unmodified SL RNA (Fig. 6, lanes 3 and 5). This is similar to what we previously observed in T. brucei (lane 1, and (14) ) and indicates that in a proportion of the SL RNA the two pyrimidine residues of the sequence AACUAA are modified at the 2` position of the ribose. The RNase mapping in Fig. 6also generated protected fragments corresponding to the SL exon, which is diagnostic of active trans-splicing (lanes 1, 3, and 5). In all three cases, addition of Ado-Hcy to the transcription mixture only revealed unmodified SL RNA (lanes 2, 4, and 6) and the concomitant absence of the SL exon fragment demonstrated that undermethylated SL RNA is not active in trans-splicing. Taken together, these experiments strongly suggested that methylation of the SL RNA 5` end is a general requirement for trans-splicing activity of trypanosomatid SL RNAs.


Figure 6: Methylation of T. cruzi and Leishmania SL RNA is required for trans-splicing activity of the corresponding SL RNA. Radiolabeled RNA from T. brucei cells (lanes 1 and 2), L. amazonensis cells (lanes 3 and 4), and T. cruzi cells (lanes 5 and 6) were subjected to RNase protection analysis after hybridization to antisense RNA probes complementary to each of the three SL RNAs. Note that the SL RNAs in these three organisms are of variable length: T. brucei, 140 nt; L. amazonensis, 96 nt; T. cruzi, 110 nt. The protected fragments were separated on a 6% polyacrylamide, 7 M urea gel. RNA from control cells is shown in lanes 1, 3, and 5; RNA from cells incubated with Ado-Hcy is shown in lanes 2, 4, and 6. The fragments corresponding to modified (m) and unmodified (u) SL RNA are indicated. The SL exon fragment is derived from trans-spliced mRNA and is diagnostic of active trans-splicing.



Since the cap 4 structure of the SL RNA is identical in T. brucei and C. fasciculata(7) , we next examined whether the mechanism of cap modification is similar in these two organisms (Fig. 7). As a source of proteins we prepared nuclear and cytoplasmic extracts from C. fasciculata and also used an available whole cell extract from L. amazonensis. When undermodified T. brucei RNPs were incubated in these heterologous extracts we detected a T2-resistant fragment that comigrated with the one originating from the homologous in vitro reaction (Fig. 7). Thus, it appeared that trypanosomatid protozoa share a common machinery for the modification of the SL RNA cap structure.


Figure 7: In vitro modification of the T. brucei SL RNA in Crithidia and L. amazonensis cell-free extracts. Undermodified P-labeled RNPs from T. brucei were incubated without extract (lane 1), or in four different extract preparations: 5 or 10 µl of a C. fasciculata nuclear extract (lanes 2 and 3); 5 or 10 µl of a C. fasciculata cytoplasmic extract (lanes 4 and 5); 5 or 10 µl of an L. amazonensis whole-cell extract (lanes 6 and 7); 5 or 10 µl of a T. brucei nuclear extract (lanes 8 and 9). The position of the T2-resistant fragment (T2R) is indicated.




DISCUSSION

In this report we describe the development of an in vitro system for the modification of the cap structure of T. brucei SL RNA. Additionally, we show that cell-free extracts from two related trypanosomatid protozoa, namely C. fasciculata and L. amazonensis, are capable of modifying the 5` end of the T. brucei SL RNA.

One interesting aspect of this in vitro system is that only the core SL RNP and not deproteinized SL RNA was utilized as a substrate for cap modification. This is similar to what has been described for pseudouridine modification (20) in the case of human U5 snRNP and for m(3)G cap hypermethylation of human U1 snRNP (21) . One explanation for this finding is that one or more of the enzymes responsible for cap 4 biosynthesis recognize one of the SL RNA-specific proteins and this would account for the specificity of the reaction. Indeed, m(3)G cap hypermethylation of human U1 snRNA requires a binding site on the Sm core domain(21) . Another not mutually exclusive possibility is that a structural determinant or a specific sequence at the 5` end of the SL RNA only becomes accessible when the SL RNA is complexed with proteins. In support of this hypothesis is the finding that the 5` end of the SL RNA is accessible to complementary deoxyoligonucleotides and RNase H only when in the RNP, but not in naked RNA. In the case of U6 snRNA it is well established that synthesis of the -monomethyl phosphate cap requires a defined RNA determinant(22) . Experiments are in progress to define the substrate requirements for SL cap 4 biosynthesis.

We have now extended our previous observation that methylation of the SL 5` end is essential for trans-splicing activity of the SL RNA to two other members of the family Trypanosomatidae, namely T. cruzi and L. amazonensis. Although a detailed structural analysis of the cap 4 has only been obtained for C. fasciculata and T. brucei, the available evidence strongly supports the view that the structure and function of the SL 5` end is common to all trypanosomatid protozoa. At present, however, the function of the SL cap 4 modifications in the trans-splicing pathway is still unclear. What we have learned so far is that the SL cap 4 modifications are not required for: (i) stabilizing the SL RNA (14) ; (ii) assembly of the salt resistant core SL RNP(14) ; (iii) establishing a specific secondary structure(23) . We have previously discussed the possibility that the SL modifications could be part of a bipartite nuclear location signal, analogous to the trimethylguanosine cap structure/Sm core domain signal for nuclear targeting of vertebrate U-snRNPs(24) . Although we find this possibility unattractive because of the rapid kinetics of utilization of the SL RNA in trans-splicing(17, 25) , at present we cannot discount it. A more attractive possibility is that the cap 4 structure or part thereof could be directly recognized by some component of the splicing apparatus. In support of this possibility are recent experiments in HeLa cells describing the identification of a nuclear cap-binding protein complex which appears to play a role in pre-mRNA recognition and whose depletion from extracts leads to inhibition of cis-splicing(26) . The availability of biochemical methods for the purification of large quantities of the SL T2-resistant structure should aid the identification of such binding activities.

At present we do not know how many different enzymes are involved in the modification of the T. brucei cap 4 structure. We anticipate that, not including the synthesis of the m^7G cap, two different types of enzymatic activities must participate in the formation of this structure: a minimum of three and possibly four 2`-O-methylases and two unique methyltransferases, one catalyzing the addition of two methyl groups to the N^6 position of the first adenosine residue, and the other specific for the addition of one methyl group to the N^3 group of the uridine at position 4 in the SL RNA. These various activities could be encoded by separate molecules or perhaps some of the enzymes have multiple activities. It could also be possible that the various enzymatic activities are in a macromolecular complex. Preliminary fractionation experiments using ion-exchange chromatography and glycerol gradients are consistent with the latter possibility.

The development of an in vitro system for cap 4 modification is a significant step toward the detailed biochemical characterization of the SL-specific methyltransferases. Analysis of these enzymes both from a molecular and cellular biology point of view will further our understanding of the biochemistry and function of RNA methyltransferases, a class of enzymes of which little is known at present. Lastly, the in vitro system will obviously prove invaluable for testing inhibitory compounds and possibly open new avenues for pharmacological intervention against trypanosomatid protozoa as a whole.


FOOTNOTES

*
This investigation received financial support from the UNDP/WORLDBANK/WHO Special Programme for Research and Training in Tropical Diseases (TDR) (to C. T.) and National Institutes of Health Grant AI28798 (to E. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 203-785-7332; Fax: 203-785-3864; Christian.Tschudi{at}yale.edu.

(^1)
The abbreviations used are: m^7G, 7-methylguanosine; Ado-Hcy, S-adenosyl-L-homocysteine; nt, nucleotide(s); RNase, ribonuclease; RNP, ribonucleoprotein particle; SL, spliced leader; snRNA, small nuclear RNA.

(^2)
E. Ullu and C. Tschudi, unpublished data.

(^3)
E. Ullu and C. Tschudi, unpublished observations.


ACKNOWLEDGEMENTS

We thank Diane MacMahon-Pratt and Peter Kima for providing the Leishmania cells, Norma Andrews for the T. cruzi cells, Philippe Male for photography, and Sandy Wolin for helpful comments on the manuscript.


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