From the Department of Microbiology and Immunology,
UCLA School of Medicine, Los Angeles, California 90095 and the
¶ Department of Medicine, West Los Angeles Veterans Affairs
Medical Center, Los Angeles, California 90073
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ABSTRACT |
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Every kinetoplastid mRNA receives a common, conserved leader sequence via the process of trans-splicing. In Leishmania tarentolae the precursor spliced leader RNA is 96 nucleotides, with a 39-nucleotide exon that is 7meG-capped and methylated on the first 4 nucleotides. trans-Splicing was inferred from the presence of tagged leader in the high molecular weight RNA population and confirmed for accuracy by cDNA cloning. Linker scan substitutions within the exon between positions 10 and 39 did not affect trans-splicing. The trans-splicing efficiency for three of the scan exons was proportional to the tagged:wild type ratio in the spliced leader precursor population. Two scan leader RNAs that were efficiently spliced showed reduced methylation. Longer exons showed reduced splicing, whereas 10- or 20-base pair deletions abolished splicing. These results indicate that size, but not content, of the exon is a constraint on the splicing process. These results, in combination with previous data eliminating a role in transcription initiation, suggest that translation may be the selective pressure on the leader content.
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INTRODUCTION |
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The 39-nt1 spliced leader (SL) in the kinetoplastids is extensively conserved (1-3), such that PCR amplification of SL RNA genes from diverse kinetoplastids can be performed with a single set of oligonucleotide primers (4, 5). These conserved sequences are presumed to be important for one or more basic biological processes, which could include transcription, trans-splicing, and translation. In the nematode Ascaris, part of the 22-nt SL sequence functions as a promoter (6); however, a major role for the SL in transcription initiation has been eliminated in Leishmania tarentolae and Trypanosoma brucei, which have upstream promoters (7, 8). An Ascaris SL of two nucleotides is accurately trans-spliced in vitro (9), indicating that information for splicing is not contained within the SL. By contrast, the SL sequence is important for both transcription (10) and trans-splicing (11) in some kinetoplastids.
Although the U5 small nuclear RNA may be cross-linked to exons near the splice junction (12), the roles of exons in cis-splicing are generally constrained by their protein-coding capacities. There are no such constraints apparent in trans-splicing. The kinetoplastid SL functions as a cap 4 donor (13) and as a 5'-untranslated region, allowing the possibility for an active role in trans-splicing. Several inter- and intramolecular interactions have been predicted to occur within the SL. Two small RNAs, SL-associated 1 and U5, have been demonstrated through in vivo cross-linking to interact with the SL (14, 15). Intramolecular interactions of the SL include alternation between two structural forms involving SL-SL (Form I) and SL-intron (Form II) base pairing in vitro (16) and in vivo (17) and proposed U1 and U5 functions (18).
To determine the importance of the conserved kinetoplastid SL in trans-splicing we evaluated several SL mutants for trans-splicing in vivo. We show that SLs containing 10-bp replacement mutations between positions 10 and 39 are efficiently and accurately trans-spliced. In addition, some spliced mutants are undermethylated at their 5' ends. These results indicate that neither the primary sequence of a 39-nt SL nor cap 4 methylation are critical for trans-splicing; however, there may be constraints on the absolute size of the SL.
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EXPERIMENTAL PROCEDURES |
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Generation of Mutations and Transfectants--
The intron tag
(IT) containing SL mutations (1/9, 10/19, 20/29, and 30/39, which
introduced an XhoI site) were generated originally for
promoter localization studies (7). An SL reporter lacking the IT was
created by removal of the IT from 30/39 and called tagged SL (tSL).
Control plasmids that lack active promoters were constructed by PCR
(7), resulting in the 67/
58 versions of 1/9, 10/19, 20/29, 30/39,
and tSL. The shortened SLs,
20 and
10, were created by subcloning
the XhoI-KpnI fragments from 10/19 or from 20/29
into the tSL vector. SLs of increased size were generated by digestion
of tSL with XhoI followed by Klenow fill-in and religation
(+4) or insertion of a double-stranded oligonucleotide possessing
XhoI-compatible ends (+24).
RNA Analysis-- RNA was purified using TriZOL reagent (Life Technologies, Inc.) and was electrophoresed through 1.1% agarose-formaldehyde or 8% acrylamide/8 M urea gels, transferred, and hybridized as described previously (7, 19). Quantitation was performed using a PhosphorImager (Molecular Dynamics).
Oligonucleotide probes used for SL RNA detection were: 10/19 tag, 5'-TGATTCCTCG AGGGCG; 20/29 tag, 5'-GTATTCCTCG AGGTAC; 30/39 tag, 5'-ACTTCCTCGA GGCTGAA; LtSLintron, 5'-GTTCCGGAAG TTTCGCATAC; +4 tag, 5'-TTCCTCGATC GAGGCT; +24 tag, 5'-TCGACTGCGA CTGGGAGTGC AGGG. Primer extension mapping of the cap 4 structure (20, 21) was performed as described (22) using oligonucleotide M838 Jr, 5'-AGCCTTGTGG GCCAGTG.RT-PCR trans-Splicing Assay--
Complementary oligonucleotides
were hybridized to the arl or ubi mRNAs and
extended by Moloney murine leukemia virus-reverse transcriptase (RT) to
produce templates for PCR analysis (RT-PCR) (22) with a second,
SL-specific oligonucleotide. The oligonucleotides used had the
following sequence: arl()68, 5'-TGCGGATCGCCTTCTGGCCACC; arl(
)36,
5'-GTAGGGTCCT CGTCGGACAGC; LtSL5'RI, 5'-GGGAATTCGCTTTCAACTAACGCTAT; ubi1, 5'-GCCTCAGCGTCTTCACGAAGATCT G; 1/9-5'RI,
5'-GGAATTCGCTTTCCTCGAGGAATAT; 10/19-5'RI,
5'-GGGAATTCGCTTTCAACTAACGCCCTC; 30/39-5'HI,
5'-GGGATCCTGTATCAGTTTCAGCCT. Products were isolated, cloned, and
sequenced as described previously (3).
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RESULTS AND DISCUSSION |
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The importance in trans-splicing of particular SL sequence elements was examined for a series of SL mutations generated following a linker scan approach in conjunction with an IT (7) and derivative mutations lacking the IT and varying in SL size (summarized in Fig. 1A). The predicted size differences because of the presence of IT sequences or exon mutations are indicated (Fig. 1A) and shown in total RNA from transfectants electrophoresed through an acrylamide-urea gel (Fig. 1B). The +4 and +24 SL RNAs each required specific exon probes and migrated as predicted on comparable 8% gels (data not shown). These data confirmed the transcript size predictions of the various constructions and demonstrated that transcription was not affected by mutation of the SL RNA gene sequences.
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To assay for trans-splicing, total RNAs from mutants were
hybridized with the exon tag oligonucleotides (Fig.
2A; data not shown for SL size
mutants and not determined for 1/9). The unspliced, mutated SL RNAs
were visible at the bottom of the blots. Hybridization of tag-specific
oligonucleotides to higher molecular weight RNA species in 10/19,
20/29, 30/39, and tSL but not in their respective promoter knockout (7)
mutants indicated that active trans-splicing of the mutated
SLs occurred. The ~1.4-kilobase bands and faint background present in
RNA from the four promoter knockout mutants were anticipated artifacts,
reflecting transcripts from the randomly initiated run-around
transcription that drives the neomycin resistance gene (23, 24). The
10,
20, +4, and +24 SLs did not appear to be efficient substrates
for trans-splicing by this assay (data not shown).
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The transfectant mRNAs were examined in a more sensitive manner and
for accuracy of splicing by RT-PCR, blot and nucleotide sequence
analysis for the presence of mutated SL on arl and
ubi mRNAs. Spliced products containing SL transcribed
from the tSL, 10, 20/29, 30/39, +4, and +24 constructions were
amplified by RT-PCR using general 5' oligonucleotides (i.e.
both WT and exon-tagged trans-splicing products amplify);
20, 1/9, and 10/19 required specific, exon tag-inclusive, 5'
oligonucleotides because of the close proximity of the tag to the 5'
end of the SL (Fig. 2B). RNAs from the inactive promoter
transfectants (
67/
58+1/9, etc.) were used as substrates in parallel
amplifications as controls for reaction specificity for the scan
mutations (data not shown). General amplification products were
transferred and hybridized with oligonucleotides directed against the
exon tag sequences, revealing trans-splicing of the queried
mRNAs by 20/29, 30/39, and tSL (Fig. 2B). Because of the
close proximity of the mutations to the 5' end of the SL, the 1/9 and
10/19 transfectants were assayed by amplification with
mutation-specific primers that indicated splicing of 10/19 but not of
1/9 (Fig. 2B). When subjected to the RT-PCR assay, the
20
and
10 SLs did not yield trans-splicing products even with
mutation-specific 5' primers (data not shown). +4 and +24 were
inefficient substrates for trans-splicing, because products
were generated with the mutant-specific 5' primer but not the general
primer (Fig. 2B).
The resulting RT-PCR products for arl and ubi mRNAs were cloned and sequenced to determine the accuracy of trans-splicing for 10/19, 20/29, 30/39, and tSL transfectants. In each case, trans-splicing with the mutated SL was mapped to the site previously identified as WT (25),2 with an additional, alternative, splice acceptor site mapped for the arl mRNA with one of the 20/29 clones (data not shown). The sequence data confirm that the RT-PCR reactions were not producing artifactual products and that these scan SLs were trans-spliced accurately.
Lack of detectable spliced products containing the 1/9 SL may be a
result of the low abundance of mutated precursor SL RNA, which was
previously shown to be efficiently transcribed but unstable (7).
Changing the initiation nucleotide from an A to a C residue will
prevent addition of the 7meG cap to the SL RNA, thus reducing the
stability of the 1/9 SL RNA and any mRNAs receiving this mutated SL. The inefficient splicing of the SL size exons +4 and +24 and the
apparent lack of splicing in 10 and
20 indicate that the size of
the SL is important. However, accurate splicing of the three scan 39-nt
SLs shows that the primary sequence outside of the cap 4 region is not
critical for trans-splicing.
The efficiencies of trans-splicing for the mutated SLs from 20/29, 30/39, and tSL transfectants were determined by comparing the proportion of mutated versus WT spliced products in the general RT-PCR product pool to the proportion of mutated versus WT SLs in the substrate populations. Direct comparison of hybridization signals for mutated and WT SLs (Fig. 3A) revealed that the 10/19 SL RNA was 2% of the total unspliced SL population, the 20/29 SL was 3%, and the 30/39 SL was 6%. tSL was approximately 2% in the RNA samples used for the PCR assays, as determined by normalization of duplicate blots with the SL-associated 1 RNA (Ref. 26 and data not shown).
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The ratio of WT:mutated SL trans-spliced products gave internally consistent results, indicating that the proportion of trans-spliced mutated:WT SLs was equivalent to the proportion of substrate mutant:WT SL in total RNA. If the mutant SL was PCR-amplified proportionally to the WT SL RNAs, we define this as efficient trans-splicing. The proportional nature of the amplification was demonstrated by amplifying varying amounts of the cloned WT and tSL spliced arl cDNAs and comparing the signals of the resulting products to the signal of the input plasmids (data not shown). Thus, the mutations within the SL had a minimal effect on the efficiency of trans-splicing.
Primer extension analysis was performed to examine the methylation
status of the cap 4 at the 5' end of the mutated SLs (Fig. 3B). The IT mutant was used for determination of WT
methylation levels; this construction has no exon mutation and showed
comparable methylation to WT SL (data not shown). 30/39 SL methylation
varied with RNA preparations between 33 (Fig. 3B) and 75%
(data not shown) as seen in IT (Fig. 3B). Surprisingly, the
unstable SL 1/9 was methylated at 75% (extended exposure, Fig.
3B), indicating that methylation and capping are not linked.
10/19 and 20/29 showed sharply reduced methylation (<5%; extended
exposure, Fig. 3B), allowing the RT to progress 3 or 4 nucleotides farther along the transcript and resulting in a larger
product. The majority of 10/19 products extended to +1; 20/29 extended
predominantly to +2. SL tag-specific oligonucleotides were used to
confirm the methylation results for the 10/19 and 20/29 transfectants
(data not shown) and to examine the 10 and
20 SL RNAs. The tSL
mutant is consistently methylated at 75% in the WT pattern, but
10
and
20 showed reduced methylation (data not shown). The mutant
phenotypes are summarized in Table I.
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Efficient splicing of under-methylated SL contrasts with findings in whole and permeabilized cells (27-29). Inhibitor studies using S-adenosyl-L-homocysteine (27) and sinefungin (28) are likely to have had a global effect on cellular methylation processes and not inhibited SL methylation exclusively. Thus, these results are reflective of the importance of methylation in the trans-splicing pathway as a whole rather than methylation of the SL in particular. The antisense oligonucleotide studies may have been confounded by the presence of the oligonucleotide itself forming a duplex structure that could affect other SL interactions in addition to disrupting the cap 4 formation process (29).
The results presented here indicate that the SL sequence between positions 10 and 39 is not important for trans-splicing and suggest that the secondary structure of stem-loop I (16) is not necessary for accurate trans-splicing. However, there appears to be an optimal size for the SL for splicing in vivo, unlike the situation in Ascaris where the exon size can vary from 2-246 nt and maintain efficient splicing in vitro (9).
Our results contrast with those of Lücke et al. (11), whose studies conclude that much of the SL sequence is important for splicing; they also conclude that size is not limiting based on a single efficiently spliced mutant with a 45-nt exon (+6 nt). The results of our two studies are largely in agreement regarding the areas of the exon important for methylation of the cap 4 (11); however, the role of the cap 4 methylations in the trans-splicing process is challenged by the efficient splicing in Leishmania of the 20/29 mutation and, based on the total RNA analysis, the 10/19 mutation. The interpretation of negative trans-splicing results must be approached with caution, because the result may be due to secondary effects of the mutation; a positive result provokes further study.
Because our studies reveal that contingencies for splicing do not exist within the primary sequence of the kinetoplastid SL, we are left to hypothesize that the conservation is necessary for proper function of the translation machinery in these organisms. In future experiments we will attempt to address this possibility directly.
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ACKNOWLEDGEMENTS |
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We thank T. Guy Roberts and Michael C. Yu for stimulating discussions, Jiang ZhiMing for making two of the constructs, and Doug Black and Dan Ray for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI34536.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Postdoctoral trainee on the Microbial Pathogenesis Training Grant 2-T32-AI-07323.
Present address: UCLA School of Dentistry, Los Angeles, CA
90095.
** To whom correspondence should be addressed. Tel: 310-825-4195; Fax: 310-206-3865; E-mail: dc{at}ucla.edu.
1 The abbreviations used are: nt, nucleotide(s); IT, intron tag; PCR, polymerase chain reaction; RT, reverse transcriptase; SL, spliced leader; tSL, tagged SL; WT, wild type; bp, base pair(s).
2 N. R. Sturm and D. A. Campbell, unpublished data.
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REFERENCES |
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