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J Biol Chem, Vol. 274, Issue 27, 19361-19367, July 2, 1999
From the Department of Microbiology and Immunology, UCLA School of
Medicine, Los Angeles, California 90095-1747
A 39-nucleotide leader is
trans-spliced onto all trypanosome nuclear mRNAs. The
precursor spliced leader RNA was tested for trans-splicing
function in vivo by mutating the intron. We report that in
Leishmania tarentolae spliced leader RNA 5' modification is
influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of
stem-loop II was found to be important for cap 4 formation and
splicing. As in Ascaris, mutagenesis of the bulge
nucleotide in stem-loop II was detrimental to
trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore
splicing, this result contrasts the findings in the kinetoplastid
Leptomonas, where mutations that restored stem-loop II
structure supported splicing. Methylation of the cap 4 structure and
splicing was also dependent on both the Sm-binding site and the
structure of stem-loop III and was inhibited by incomplete 3' end
processing. The critical nature of the L. tarentolae
Sm-binding site is consistent with its essential role in the
Ascaris spliced leader RNA, whereas in
Leptomonas mutation of the Sm-binding site and deletion of
stem-loop III did not affect trans-splicing. A pathway for
Leishmania spliced leader RNA processing and maturation is proposed.
Kinetoplastid nuclear gene expression is dependent on the
trans-splicing process. The common substrate for all
trans-splicing reactions is the spliced leader
(SL)1 RNA, also known as the
mini-exon derived RNA, whose first 39 nt constitute the 5' ends of both
mono- and polycistronically synthesized mRNAs (1). The
polycistronic pre-mRNAs require trans-splicing to
acquire the specialized "cap 4" structure on the SL RNA. The cap 4 consists of a 7mG attached to the first nucleotide (2), in
addition to methylation of the first four and sixth nucleotides of the
SL RNA (3-5). These modifications are made to the primary SL RNA and
spliced onto the mRNA as part of the 39-nt exon. The cap 4 may have
roles in mRNA trans-splicing, transport, stability and translation.
The SL RNA contains two functional domains as follows: the exon and the
intron or snRNA-like domain (6). The exon sequence is conserved among
38 different members of the order Kinetoplastida (7). Positions 1-9
and 20-39 of the exon are nearly identical, whereas positions 10-19
are relatively heterogeneous and characteristically A/T-rich. This
conservation cannot be ascribed to internal promoter location in
Leishmania (8, 9), as found in Ascaris (10). It
was surprising that mutations within positions 20-39 permitted accurate trans-splicing in Leishmania tarentolae
and did not lower splicing efficiency (11) because these results
contrasted with findings in Leptomonas (12). Thus, the
results in L. tarentolae more closely resemble the findings
in worms as follows: in Ascaris, exon sequences are not
necessary for trans-splicing in vitro (13); in
Caenorhabditis elegans, length, primary sequence, and
composition of the SL are not critical parameters for essential
embryonic function, although certain nucleotides may be essential for
in vivo splicing of the SL1 RNA (14, 15).
The primary sequence of the SL RNA intron is not conserved among the
trypanosomatids (7); however, the secondary structure is consistent
(16). This structure has been confirmed by physical-chemical and
enzymatic studies (17, 18) and examined by mutagenesis (12). An
equivalent structure is also conserved in the nematode SL RNAs (16,
19). The intron contains a putative Sm-binding site (16), an element
found in the small nuclear RNAs of higher eukaryotes but apparently
lacking in all U-RNAs of kinetoplastids (20) except U5 RNA (21, 22).
The Sm-binding element is required for SL RNA trans-splicing
in Ascaris (23) but not in Leptomonas (12).
We demonstrated recently that the T tract downstream of the SL RNA gene
is a transcription termination element and that staggered T tract
termination products are processed via nucleolytic cleavage to the base
of stem-loop III (24). The signals for 3'-processing begin in the
Sm-binding site at position 76 and include the structure, but not
content, of stem-loop III. Studies in Leptomonas seymouri demonstrated that mutation of a variety of elements in the intron was
acceptable for trans-splicing (12), whereas in
Leptomonas collosoma the loop portions of stem-loops II and
III were tolerant to insertions but not to replacement with the
Trypanosoma brucei intron (25). By contrast, the bulge of
stem-loop II was critical for trans-splicing in
Ascaris (26).
In this paper we report that methylation of nucleotides in the cap 4 structure of the Leishmania SL RNA is influenced by
formation of stem-loop III, the Sm-binding site, and specific sequences in stem-loop II. The methylation of the cap 4 structure correlates with
correct 3' end formation; defects in 3' end processing and cap 4 formation result in failure of the mutated SL RNA to undergo trans-splicing. However, correct maturation of the SL RNA is
not sufficient to obtain a positive splicing phenotype since mutation of nucleotides in the stem I region of the intron can also result in
loss of function. Our data from L. tarentolae broadly
reflect the results obtained in vitro in the nematode
Ascaris, where nucleotides in stem-loop II and the
Sm-binding site are necessary for splicing. Our data broadly contrast
the results obtained in Leptomonas, where the structure and
not the primary sequence of stem-loop II was necessary for splicing,
and where the Sm-binding site and stem-loop III were not required for
splicing. A model summarizing the features of the Leishmania
SL RNA involved in maturation and trans-splicing is presented.
Generation of Mutations and Transfectants--
Mutagenesis was
performed using the Sculptor Mutagenesis kit (Amersham Pharmacia
Biotech) or using PCR to generate mutagenized DNA fragments for
subcloning into the transfection vector. Mutated fragments were cloned
for transfection into a pX plasmid (27) containing an SL RNA gene
(9).
Transfections were performed by electroporation as described (11).
Nucleic Acid Isolation and Gel Analysis--
RNA was purified
using TriZOL reagent (Life Technologies, Inc.) and was electrophoresed
through 1.1% agarose-formaldehyde, blotted, and hybridized as
described previously (28). Quantitation was performed using a
PhosphorImager (Molecular Dynamics).
Reverse Transcriptase-PCR trans-Splicing Assay--
To assay for
trans-splicing of mutant-SL RNAs, complementary
oligonucleotides were hybridized to Arl mRNA (29) and extended by
Moloney murine leukemia virus-reverse transcriptase (RT) to produce
templates for PCR analysis (RT-PCR (30)) with a second, SL-specific
oligonucleotide. The following oligonucleotides were used: Arl( 5' End Analyses--
5' cap 4 methylation was assayed by primer
extension of total RNA with an exon tag-specific oligonucleotide
(28/39-tag) as described previously (9, 11).
Intron Mutations Affect trans-Splicing--
To localize specific
elements within the intron of the SL RNA that play a role in the
process of trans-splicing, a systematic mutagenesis approach
was adopted (Fig. 1). To differentiate
mutated, episomally derived SL RNA from the endogenous WT SL RNA
population, an exon tagged at positions 28 and 30-39 (28/39), which
was previously shown to trans-splice accurately and
efficiently (11), was used as a molecular tag (tSL RNA) for
detection by hybridization. A series of linker scan (CTCGAGCTCA)
mutations in the tSL RNA gene was created for transfection to L. tarentolae. Two mutations in the 40-49 region were created as
follows: a mutant with alterations in bases 43 and 44 (43/44 = GT
versus TG in WT) tested a postulated SL RNA-U6 snRNA
interaction (31), and a second mutant, altered at positions 42-48
(42/48), changed all but the splice donor site with the linker scan
sequences. Subsequent intron mutations continued from position 50 (52/59) and proceeded through the end of the intron. Three mutations
lay downstream of the mature 3' end of the SL RNA transcript (position
96) and were included to identify potential adjacent expression
elements (100/109, 110/119, 120/129; WT sequence not shown).
Analysis of total RNA from the transfectants demonstrated tSL RNA
(~96 nt) in all the samples by low resolution formaldehyde-agarose gel blotting (Fig. 2A). A
broadened size range from the wild-type (WT) 96 nt to at least 175 nt
was noted for the 100/109 tSL RNA, consistent with discrete higher
molecular weight bands visible in higher resolution gel analyses (24).
The presence of tagged precursor SL RNA indicated that all the mutants
have the potential to trans-splice. An artifactual
transcript (~1.45 kb) that accumulated in each sample provided an
internal control for transfection, should stability be disrupted. The
presence of the exon tag in a range of high molecular weight RNA
species (500 nt to 9 kb) in the tSL, 43/44, 100/109, 110/119, and
120/129 samples suggested that active trans-splicing was
occurring in these transfectants (11). Splicing of 43/44 is consistent
with results from a similar study in Leptomonas (12).
Conversely, splicing of the tagged exon was impaired in the 42/48,
52/59, 62/69, 70/79, 80/89, and 90/99 mutants, where only substrate tSL
RNA and the artifactual transcripts accumulated. The levels of
accumulating tSL RNAs varied relative to the artifactual transcripts
and the episomally encoded drug selectable marker mRNA NEO (data
not shown). The 52/59 mutant in particular showed an increased
accumulation of substrate molecules relative to other non-splicing
mutants.
In addition, trans-splicing was assayed by RT-PCR (11, 29)
to detect low levels of splicing (Fig. 2B). When "total"
SL primer (i.e. will amplify from both WT and tSL exons) was
used in the amplification, all samples showed the positive control WT
amplification products, but the 28/39-tag oligonucleotide hybridized only to the tSL, 43/44, 100/109, 110/119, and 120/129 products, consistent with the total RNA blot analysis. However, using a tSL-specific primer for amplification, some level of splicing was
detected in all but the 70/79 mutant. These experiments included WT
L. tarentolae RNA, no reverse transcriptase, and no RNA
reactions as negative controls for contamination and dependence on the
use of RNA templates. Furthermore, a promoter knockout in combination with tSL (
Structural analyses of the SL RNA predict three stem-loop structures
and a single-stranded region containing the Sm-binding site (Ref. 17;
Fig. 1). Previously, it was demonstrated that stem-loop I is not
required for trans-splicing in L. tarentolae (11). Because trans-splicing was reduced or abolished in
mutants 52/59, 62/69, 70/79, 80/89, and 90/99, we considered the
effects of mutations in stem-loop II, the Sm-binding site, and
stem-loop III on SL RNA trans-splicing with regard to the
structural or sequence elements. The mutations described below are
organized with regard to both these elements and the linker scan
mutation results in the following order: structural features of
stem-loop II, fine analysis of the 70-81 region which includes part of
stem-loop II and the Sm-binding site, and features of stem-loop III.
Sequence and Structure of Stem-Loop II Are Necessary for
trans-Splicing--
Two mutations, 52/59 and 62/69, disrupted
stem-loop II (Fig. 3A) and
were not efficiently trans-spliced (Fig. 2). To address the
importance of stem-loop II, 52/59 was further mutated to restore base
pairing (52/59 + 65/72; Fig. 3A); this replaced the stem structure but with a different sequence content than WT. A further mutation was designed (42/48 + 77/80) to restore a possible extension of stem-loop II in the 45-48 region, which was disrupted by mutations 42/48 and 70/79 (Fig. 3A). 77/80 was also tested for
independent effects due to its disruption of the conserved Sm-binding
site. Neither of the compensatory base pairing mutations restored
trans-splicing (Fig. 3B). 77/80 alone or in
combination with 42/48 resulted in extended, heterogeneous 3' end
formation (data not shown) consistent with the 70/79 phenotype (24),
whereas 52/59 and 52/59 + 65/72 possessed correct 3' ends (data not
shown). 42/48 + 77/80, 52/59 + 65/72, and 77/80 showed undermethylated
cap 4 structures (data not shown), as did 42/48, 52/59, and 70/79
(24).
Both the structure and sequence content of stem-loop II are thus
important features in the maturation of the SL RNA precursor. The
structure alone is not sufficient to direct either cap 4 methylation or
splicing. The stem-loop II extension structure may play an intermediate
role in the splicing pathway, but it is not sufficient to restore
processing or splicing.
The Stem-Loop II Single Nucleotide Bulge and Sm-binding Site Affect
trans-Splicing and Cap 4 Methylation--
Because 70/79 altered most
of the Sm-binding site and resulted in no trans-splicing and
defects in both 5' and 3' end formation, we examined the area in finer
detail. A 2-bp transversion (TV) series was created from position
70 to 81; in addition, 70/79 and 75/81 TV mutations were made (Fig.
4A). It should be noted that
70/71 and 72/73 comprise part of stem-loop II (see Fig. 1) and that
74/75 TV may extend the Sm sequence (AAUCUUUUGG).
The total RNA of these transfectants revealed a variety of phenotypes
for trans-splicing and methylation. By formaldehyde-agarose gel analysis, only the 74/75-tSL RNA was an efficient
trans-splicing substrate, with low levels of splicing
evident in 72/73 (Fig. 4B). The presence of tSL RNA but lack
of the 1.45-kb artifact RNA in mutants 76/77, 70/79 TV, and 75/81
suggested additional increased stability phenotypes. Primer extension
revealed an intriguing gradient of SL RNA cap 4 methylation in the
70/71, 72/73, and 74/75 mutants (Fig. 4C), which showed low
(5%), medium (40%), and normal (75%) methylation, respectively, and
were trans-spliced proportional to their methylation state.
Thus, as a component of the Sm-binding consensus, A75 does
not appear to be an essential nucleotide in Leishmania; alternatively, the 74/75 mutation is a biologically acceptable extension of the Sm site.
The Stem-Loop III Formation Affects trans-Splicing and Cap 4 Methylation--
Mutations 80/89 and 90/99 disrupted stem-loop III
(Fig. 5A) and resulted in
3'-extended, undermethylated tSL RNAs (11) that were not efficiently
trans-spliced. To examine further the role of stem-loop III
structure, a series of mutations was created that disrupted and then
replaced 1 or 3 bp of the stem and that altered the loop sequence (Fig.
5A). The single base disruptions were expected to disrupt
only one rung of the stem, and thus lead to a minor size difference in
the tSL RNA, whereas the triple base mutations were designed to disrupt
the stem completely.
Formaldehyde-agarose gel analysis of total RNA showed that the single
base pair-disrupted SL RNAs (83, 96) could be trans-spliced, but the triple base pair-disrupted SL RNAs (83/85, 94/96) were not
trans-spliced efficiently (Fig. 5B). The base
pairing compensated SL RNAs (83 + 96, 83/85 + 94/96) were both
efficient splicing substrates. The 88/91 loop mutation SL RNA
appeared to be spliced with lowered efficiency based on the reduced
mRNA smear relative to the tSL RNA substrate levels. These
experiments show that the structure, but not the primary sequence, of
stem-loop III is necessary for trans-splicing.
Primer extension analysis indicated that the cap 4 methylation patterns
(Fig. 5C) correlated with the levels of
trans-splicing. 83, 96, and 83 + 96 were methylated
efficiently and trans-spliced relative to the abundance of
free tSL RNA (Fig. 5B) and by total SL RT-PCR assays; 83/85
and 94/96 showed less than 5% methylation and had splicing that was
only detectable by the mutation-specific RT-PCR assay (data not shown).
In mutant 83/85 + 94/96, the tSL RNA cap 4 was methylated to WT levels,
thus the compensating mutations, which restored stem-loop III, also
restored a structural signal for the cap 4 methylase. 88/91 showed
approximately 50% methylation (Fig. 5C) and displayed
reduced splicing (Fig. 5B); the intron tag previously used
to follow SL RNA transcription (9) was inserted into this loop and does
not interfere with cap 4 methylation of tagged SL RNA (11). Thus,
methylation is linked to the formation of a minimum of 4/5 bp stem in
stem-loop III.
We have made a series of mutations in the region downstream of the
exon in the SL RNA gene to examine effects on
trans-splicing. We have assayed for the ability of mutated
SL RNA to trans-splice, and we have correlated this with
correct cap 4 formation, transcription termination, and 3' end
processing as determined here and elsewhere (11, 24). A summary of nine
phenotypes associated with the intron mutations is presented in Table
I. In the majority of mutants, reduction
or loss of trans-splicing correlates with defects in cap 4 methylation and 3' end maturation. Where cap 4 methylation and 3' end
formation are WT, primary catalytic elements may have been mutated
(e.g. 42/48).
The mutant phenotypes have allowed us to evaluate structures and
elements that may be important for SL RNA maturation and to propose a
possible pathway for discrete processing steps in L. tarentolae (Fig. 6). In this model,
the T tract functions as a transcription termination element (24). The
Sm-binding site and stem-loop III structure are required to allow
precise 3' end maturation. Formation of the mature 3' end, along with
elements within stem-loop II, are required for cap 4 synthesis, as is
the 10-29 region of the exon (11). Nucleotides within the intron region of stem I are likely to be required for splicing catalysis.
The Role of Intron Structures in trans-Splicing and
Cap 4 Formation for the Leishmania Spliced Leader RNA*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)68,
5'-TGCGGATCGCCTTCTGGCCACC; LtSL5'RI, 5'-GGGAATTCGCTTTCAACTAACGCTAT; 30/39-5'HI, 5'-GGGATCCTGTATCAGTTTCAGCCT. Amplification products were
run in 1.5% agarose gels, denatured, blotted, and hybridized with
28/39-tag oligonucleotide (5'-ACTTCCTCGAGGCTGAA) to detect the exon tag
or wild-type (5'-CAATAAAGTACAGAAACTGA) exon sequence (corresponding to
positions 20-39). The expected product size is ~300 bp.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A linker scan series was generated through
the intron of the L. tarentolae SL RNA gene. The
secondary structure of L. tarentolae SL RNA is based on the
L. collosoma structure that predominates in vivo
(17, 18); numbering is relative to the start of transcription. The
stem-loop structures are labeled I, II, and III,
and the exon-intron junction is indicated by the arrow after
nucleotide 39. The Sm-binding site sequence AUUUUGG and the mature 3'
end of the SL RNA are indicated. The 7mG cap is shown at
the 5' end, along with the methylated nucleotides (*) that comprise the
cap 4 structure (3, 4). All constructs contained an exon tag (tSL)
sequence at position 28-39, indicated by lowercase letters.
The scan sequence, CTCGAGCTCA, included XhoI and
SacI sites; positions within the 10-base pair blocks that
are altered are indicated in lowercase letters adjacent to
the WT position. Three additional scan mutations, 100/109, 110/119, and
120/129, are not shown but are described elsewhere (24).
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Fig. 2.
SL RNA intron mutations affect
trans-splicing. A, transcription and
variable trans-splicing of mutated SL RNAs. Total RNA from
WT, the tSL, and the double-site mutants (containing tSL and mutations
detailed in Fig. 1) were electrophoresed through a 1.1%
formaldehyde-agarose gel, blotted, and hybridized with an
oligonucleotide (28/39 tag) directed against the exon tag. Two of the
ribosomal RNA exclusion zones (2.2 and 1.5 kb) and the SL RNA (96 nt)
served as size markers (M). B, low levels of
trans-splicing detected by RT-PCR. RT-PCR assays were
performed on the mutant RNA populations with either nonspecific SL RNA
primer (top and middle) or 28/32 tag-specific SL
RNA primer (bottom) using the L. tarentolae Arl
mRNA (29) as the query template. Control reactions included RNA
from a mutant containing tSL coupled with an inactive promoter,
67/58 + tSL (11), -RT, and -RNA
reactions. Additional 5' and 3' end phenotypes (24) are summarized as
follows: + = WT, ± = WT, and = T tract for 3' end formation,
and + = WT, ± = 50%, and = < 5% for cap 4 methylation.
67/58 + tSL (11)) was used as a control for spurious PCR
amplification; this cell line resulted in an artifactual ~1.45-kb transcript containing the 28/39-tag sequence but no mature tSL RNA
(shown in Fig. 5B) and did not yield a tSL PCR product.
Previously determined phenotypes for cap 4 and 3' end formation (24)
are also indicated in Fig. 2. Thus, trans-splicing was
adversely affected in mutants 42/48, 52/59, 62/69, 80/89, and 90/99 and
appeared to be abolished in mutant 70/79.
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Fig. 3.
Stem-loop II structure and primary sequence
are necessary for trans-splicing. A,
disruptions of stem-loop II and compensatory mutations. The disruptions
in stem-loop II caused by 52/59 and 62/69 are shown, with the
structural compensatory mutation 52/59 + 65/72. All mutated positions
are boxed. B, the compensatory changes do not
rescue trans-splicing. Examination of total RNAs from the
original and compensatory mutants on a blot of a 1.1%
agarose-formaldehyde gel hybridized with the 28/39 tag oligonucleotide.
43/44 is included as a positive splicing control. Markers
(M), 3' end, and cap 4 phenotypes are as described for Fig.
2 or in the text.
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Fig. 4.
The stem-loop II bulge and Sm-binding site
affect trans-splicing and methylation.
A, mutations in the 70/81 region. Nucleotide
transversions were used to maximize structural disruption. The
Sm-binding site (black background) and stem-loop II bulge
nucleotide (oval) are indicated. B, the
trans-splicing ability of the mutants described in
A is shown in a blot of a 1.1% agarose-formaldehyde gel
hybridized with the 28/39 tag oligonucleotide. tSL is included as a
positive splicing control. Size markers (M) and 3' end
phenotypes are as described in Fig. 2. C, variable
methylation in the 70-81 mutants. Primer extension analysis was
performed. tSL is shown as a representative of wild-type cap 4 formation. The +1 to +5 RT termination sites are marked.
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Fig. 5.
Stem-loop III formation is necessary for
trans-splicing. A, the WT stem-loop
III structure and its disruption by various mutations. Single (83 and
96) and triple (83/85 and 94/96) base pair stem disruptions and their
compensatory stem structure mutations (83 + 96 and 83/85 + 94/96) are
shown. A loop transversion mutation was also created (88/91). All
mutated positions are boxed. B, single and
compensatory mutants trans-splice. A 1.1%
agarose-formaldehyde gel blot was hybridized with 28/39 tag. tSL is a
positive splicing control, and 67/58 + tSL is a negative splicing
control (11), size markers (M), and 3' end formation are as
indicated in Fig. 2. Additional 3' end phenotypes +0.5 and 1 refer to
discrete transcript size changes, and nd = not done.
C, stem-loop III disruption reduces methylation. Primer
extension analysis was performed on the stem-loop III mutant SL RNAs.
The SL RNA nucleotide positions corresponding to the product sizes are
indicated.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of mutant phenotypes
, 
5%
activity.
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Fig. 6.
A summary of SL RNA elements important for 3'
and 5' end formation and trans-splicing. The
L. tarentolae SL RNA structure shown in Fig. 1 has been
modified according to the data presented in this paper and elsewhere
(24). The 3' end of the SL RNA precursor is shown by the "staggered
termination" in the T tract, and the mature 3' end is indicated by an
arrow at the base of stem-loop III. Nucleotides that have
been examined by mutagenesis and do not disrupt normal SL RNA
maturation or splicing have been changed to "N";
proposed base pairing interactions that are not necessary are deleted.
The structures and sequences proposed to interact with maturation
components, especially for cap 4 methylation, are included in the
shaded oval and are discussed in the text.
Transcription of the SL RNA gene in vivo terminates in a T
tract of greater than six T residues (Fig. 6, step 1).
Mutation of the Sm-binding site (e.g. 76/77) and stem-loop
III (e.g. 90/99) yielded mutants with defects in the
nucleolytic formation of the mature 3' end of the SL RNA, demonstrating
a cooperative function between these two elements (step 2).
Structure rather than primary sequence of the stem-loop III stem was
required for trans-splicing. Consistent with this, an
8-nucleotide insertion in the loop of stem-loop III in L. collosoma did not affect trans-splicing (25); however,
in L. seymouri deletion of stem-loop III resulted in an
actively trans-spliced and normally methylated SL RNA (12). Mutants that do not terminate accurately due to the disruption of their
downstream T tract show an intermediate cap 4 phenotype (e.g. 100/109) that we interpret as indicative of a temporal
order of 3'-processing (step 2) followed by cap 4 methylation (step 3). Methylation alone is not sufficient to
confer splicing potential, since the 42/48 mutant is normally
methylated but a marginal trans-splicer (a phenotype similar
to the Leptomonas 
stl II mutant (12)). This suggests that
the intron region of stem I may be involved in catalytic steps of
trans-splicing (step 4). Positions 43/44 are not
implicated in the 42/48 splicing defect. Nucleotides 42 + 45-48 may
interact with other splicing entities such as SLA1 RNA (32) or U5 snRNA
(21, 22). Nucleotides +7 and +8 of the intron (equivalent to positions
46-47 in L. tarentolae) in L. collosoma can be
mutated without affecting trans-splicing (22).
The essential nature of the Sm-binding site for in vivo trans-splicing in L. tarentolae agrees with data from Ascaris, where in vitro studies showed that the Sm-binding site was required for SL RNA trans-splicing (23). We are aware of the limitations in comparing in vitro trans-splicing assays with in vivo splicing phenotypes (12). We generally interpret lack of splicing phenotypes as due to splicing catalysis or, when they are detected, to maturation-related defects, but at this level of analysis our studies cannot exclude other explanations, for example impaired nucleus-cytoplasm-nucleus shuttling of the SL RNA. In contrast, a Leptomonas Sm-binding site mutant ("sub-Sm" (12)) that closely approximated a non-splicing, 3'-extended L. tarentolae counterpart (78/79) was viable for ribonucleoprotein assembly and splicing. Splicing in the 74/75 mutant, which has a transversion of the A of the Sm site, may reflect flexibility within the conserved Sm-binding site, as found in the U5 snRNA of Saccharomyces (33). An additional experimental difference to be considered between the two studies in trypanosomatids is that the exon tag in L. tarentolae consisted of 11 mutated nucleotides, whereas that in Leptomonas consisted of one mutated nucleotide. The contradictory results for Sm-binding site and stem-loop III in the kinetoplastids may be informative in interpreting our results as follows: given that stem-loop III does not contain primary sequence necessary for trans-splicing in Leptomonas, our non-splicing phenotypes may be secondary effects (e.g. additional 3'-extended sequences may inhibit the folding of stem-loop II).
As indicated by mutant 70/71, elements in stem-loop II are required for
the intron component of cap 4 formation. In Leptomonas, deletion or substitution of stem-loop II above the bulge position did
not affect cap 4 methylation (12), suggesting that some of the
methylation phenotypes that we observed may be secondary effects due to
interference with secondary or tertiary structure formation within the
SL RNA itself or between the SL RNA and other splicing components.
Similar to L. tarentolae, nucleotides in stem-loop II of the
Ascaris SL RNA (positions 39-42 and 61-65) are essential
for trans-splicing and include a single nt (U, position 62)
bulge (26). Consistent with the L. tarentolae results and contrasting the Leptomonas results, deletion of nucleotides
59-68 in stem-loop II of the Leishmania amazonensis SL RNA
(1) resulted in either inefficient or no trans-splicing
(8).
In this study we have identified how various structures within the
intron of the SL RNA are interdependent in 3' end formation and cap 4 methylation, and we provide a possible pathway to describe the
processing steps. We distinguish among trans-splicing
negative mutants that are defective for discrete steps in SL RNA
maturation and a mutant that may be affected in catalytic steps. These
and subsequent mutants will facilitate studies on the intracellular trafficking of SL RNA, the identification of new
trans-spliceosomal proteins and protein-RNA interactions,
and allow testing of new models of interactions with other splicing
RNA/ribonucleoproteins.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Steve Beverley for the pX plasmid, T. Guy Roberts, and Michael C. Yu for stimulating discussions, and Doug Black, Larry Feldman, and Dan Ray for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part 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 supported by Microbial Pathogenesis Training
Grant 2-T32-AI-07323.
§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1747. Tel.: 310-825-4195; Fax: 310-206-3865; E-mail: dc{at}ucla.edu.
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ABBREVIATIONS |
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The abbreviations used are: SL, spliced leader; tSL, tagged SL; Arl, ADP-ribosylation factor-like; PCR, polymerase chain reaction; RT, reverse transcriptase; TV, transversion; WT, wild type; bp, base pair; kb, kilobase pair; nt, nucleotide(s); snRNA, small nuclear RNA.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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