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 (mG) ()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 mguanosine(5`)ppp(5`)-N,N,2`-O-trimethyladenosine-p-2`O-methyladenosine-p-2`-O-methylcytosine-p-3,2`-O-dimethyluridine (7) . The N,N,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 mG 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

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

Analysis of Cap 4 Structure

RESULTS

Assays for Modifications

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

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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) , ()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 (mguanosine(5`)ppp(5`)-N,N,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 mG (pmG), 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 [-P]ATP or [-P]UTP (data not shown). Since the relative mobilities of these two spots are indistinguishable from N,N,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

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

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 mG 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, mG 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 mG 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 position of the first adenosine residue, and the other specific for the addition of one methyl group to the N 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.

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

()

E. Ullu and C. Tschudi, unpublished data.

()

E. Ullu and C. Tschudi, unpublished observations.

ACKNOWLEDGEMENTS

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