(Received for publication, May 9, 1995)
From the
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.
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 m
G
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.
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.
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.
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
(m
guanosine(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 m
G (pm
G),
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.
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.
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.
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,
m
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 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.