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Molecular and Cellular Biology, February 1999, p. 1595-1604, Vol. 19, No. 2
Department of Microbiology and Immunology,
University of California Los Angeles School of Medicine, Los
Angeles, California 90095-1747
Received 16 July 1998/Returned for modification 14 October
1998/Accepted 6 November 1998
Addition of a 39-nucleotide (nt) spliced leader (SL) by
trans splicing is a basic requirement for all trypanosome
nuclear mRNAs. The SL RNA in Leishmania tarentolae is a
96-nt precursor transcript synthesized by a polymerase that resembles
polymerase II most closely. To analyze SL RNA genesis, we mutated SL
RNA intron structures and sequence elements: stem-loops II and III, the
Sm-binding site, and the downstream T tract. Using an exon-tagged SL
RNA gene, we examined the phenotypes produced by a second-site 10-bp
linker scan mutagenic series and directed mutagenesis. Here we report
that transcription is terminated by the T tract, which is common to the
3' end of all kinetoplastid SL RNA genes, and that more than six T's
are required for efficient termination in vivo. We describe mutants
whose SL RNAs end in the T tract or appear to lack efficient
termination but can generate wild-type 3' ends. Transcriptionally
active nuclear extracts show staggered products in the T tract,
directed by eight or more T's. The in vivo and in vitro data suggest
that SL RNA transcription termination is staggered in the T tract and
is followed by nucleolytic processing to generate the mature 3' end. We
show that the Sm-binding site and stem-loop III structures are
necessary for correct 3'-end formation. Thus, we have defined the
transcription termination element for the SL RNA gene. The termination
mechanism differs from that of vertebrate small nuclear RNA genes and
the SL RNA homologue in Ascaris.
The spliced leader (SL) RNA is
central to kinetoplastid nuclear gene expression. The SL RNA, or
mini-exon-derived RNA, is a primary transcript that is synthesized
independently of the pre-mRNA and trans spliced onto all
nuclear mRNAs (1), most of which are synthesized as
polycistronic precursors. SL RNA transcription represents approximately
10% of total RNA synthesis (10, 42). The approximately 100 copies of the SL RNA gene are tandemly repeated in a head-to-tail
manner in the chromosomal locus; the transcription of each gene is
directed by an upstream promoter (26, 30, 54, 62). It has
been demonstrated by nuclear run-on analysis that transcription does
not proceed into the SL RNA gene-flanking region (40, 62);
therefore, the intergenic region (256 bp of a 363-bp repeat in
Leishmania tarentolae, 1.2 kb of a 1.35-kb repeat in
Trypanosoma brucei) can be considered a nontranscribed spacer (62). The presence of some termination element near
the 3' end of the SL RNA sequence is thus indicated experimentally.
Although the 39-nucleotide (nt) SL exon is well conserved in two
domains, the primary sequence of the SL RNA intron is not conserved
among the trypanosomes (73). However the secondary structure
of the SL RNA, composed of three stem-loops and a single-stranded region containing a putative Sm-binding site (Fig.
1A), is consistent (11). This
structure has been confirmed by physical-chemical and enzymatic studies
(29, 44) and examined by mutagenesis (47). An
equivalent structure is also conserved in the nematode SL RNAs
(11, 53). The Sm-binding site (11) is an element found in the U-RNAs of higher eukaryotes but, with the exception of U5
(77), apparently lacking in the small nuclear U-RNAs of kinetoplastids (50).
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transcription Termination and 3'-End Processing of
the Spliced Leader RNA in Kinetoplastids
ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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FIG. 1.
Secondary structure of the L. tarentolae SL
RNA and linker-scanning mutations in the intron. (A) Secondary
structure of L. tarentolae SL RNA, based on the L. collosoma structure (form II) that predominates in vivo (29,
44); 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 nt 39. The Sm-binding-site
sequence AUUUUGG is indicated. The 7meG cap is shown at the 5' end,
along with the methylated nucleotides (*) that comprise the cap 4 structure (21, 55). (B) A linker-scanning series was
generated through the intron of the L. tarentolae SL RNA
gene and beyond. Numbering is relative to the start site of SL RNA
transcription. All constructs contained an exon tag (tSL) sequence at
nt 28 to 39, indicated by lowercase letters. The scan sequence,
CTCGAGCTCA, included XhoI and SacI sites;
positions within the 10-bp blocks that were not altered from WT are
indicated by dots. The mature 3' end of the SL RNA is indicated.
A poly(T) tract is a common feature of the 3' end of SL RNA genes in kinetoplastids. The poly(T) tract ranges in length from 5 T's in Leishmania donovani (18) to 31 in Trypanosoma cruzi (15). This homopolymeric motif has been postulated to be a termination element for transcription of the SL RNA genes. The ability of four T's to terminate RNA polymerase III (pol III) transcription has been used as an argument in favor of pol III transcription of SL RNA genes (24).
No pol II transcription terminators have been identified in trypanosomatids. In vertebrates, small nuclear RNA snRNA genes transcribed by pol II possess conserved downstream sequence elements that determine 3'-end formation, most probably through transcription termination (33). Although a 12-of-13 match with the vertebrate U snRNA transcription termination box was found 9 nt downstream of the SL RNA gene coding region in Ascaris (53), mutagenesis of this box and beyond did not affect SL RNA 3'-end formation (28), leading the authors to conclude that the signals for 3'-end formation lay entirely within the transcribed region and adjacent 12 nt.
The identification of the polymerase that is responsible for the
transcription of the SL RNA gene has been approached by several groups
with at least three kinetoplastids. Drug inhibition studies with
disrupted or permeabilized cells and in vitro transcription systems
support pol II transcription by the criteria of Tagetitoxin (26,
62) and Sarkosyl (60, 62) responses. Similar studies found different results with 
-amanitin (pol II [5,
41], pol III [24], and unclear
[38]) and 1,10-phenanthroline with or without
Mn2+ (pol II [61] and pol III
[24]). The presence of putative box A and box B pol
III promoter elements in the SL RNA gene has also been described;
however, it has since been shown that disruption of these elements does
not affect transcription (2, 47, 54, 62). Studies of the
K+ ion requirement support pol III transcription
(70). Because these studies were carried out with several
kinetoplastids under variable reaction conditions, it is difficult to
resolve the polymerase issue; however, in L. tarentolae,
three of four indicators tested indicated pol II activity while the
fourth was ambiguous (62).
We have recently identified the core promoter elements of the L. tarentolae SL RNA gene (80). To define SL RNA gene termination and 3'-end formation elements that might be distinct from elements related to trans splicing, we took an in vivo approach to challenge various structures and sequence elements by using stable transfection of L. tarentolae cells with exon-tagged markers. The results obtained in vivo were complemented by using an in vitro transcription assay. In this paper, we report that the SL RNA intron and poly(T) tract have distinct effects on transcript termination and 3'-end formation but do not alter transcriptional initiation. We demonstrate that transcription is terminated in the poly(T) tract and is followed by removal of 6 to 8 nt to the base of stem-loop III, which is dependent on both Sm-binding site sequences and a stem-loop III structure. Transcription termination, but not the 3'-processing activity, was achieved in transcriptionally competent L. tarentolae nuclear extracts.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Generation of mutations and transfectants. Mutagenesis of the L. tarentolae SL RNA gene was performed with the Sculptor mutagenesis kit (Amersham) or by PCR (62). Mutated fragments were cloned into the pX transfection plasmid (43). L. tarentolae transfections were performed by electroporation as described previously (66).
Nucleic acid isolation and gel analysis. RNA was purified with TriZOL reagent (Gibco/BRL) and was electrophoresed through 6% (0.4 mm thick, 40 cm long for high resolution) or 8% (1.5 mm thick, 15 cm long for medium resolution) acrylamide-8 M urea gels, blotted, and hybridized as previously described (62, 67). Quantitation was performed with a PhosphorImager (Molecular Dynamics).
5'- and 3'-end analyses. Transcript initiation was assayed by primer extension of total RNA as described previously (62, 66).
The 3'-end mapping of wild-type (WT) and mutant SL RNAs was performed by poly(A) tailing total RNA followed by reverse transcription with an oligo(dT) primer and PCR with the oligo(dT) primer and SL-specific oligonucleotide LtSL5'RI or 30/39-5'HI (66) as described previously (58).In vitro transcription assay. Nuclear extracts were prepared and in vitro transcription reactions were carried out for L. tarentolae essentially as described for T. brucei (25, 26).
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Intron mutations do not affect transcription initiation. To localize specific elements within the primary sequence or secondary structure of the SL RNA (Fig. 1A) that play a role in the maturation of the molecule, a systematic mutagenesis approach was adopted (Fig. 1B). To differentiate mutants from the endogenous SL RNA population, an exon mutated at positions 28 and 30 to 39 (28/39), which was previously shown to trans splice accurately and efficiently (66), was used as a molecular tag (tSL RNA) for detection by hybridization. A series of 10-bp linker-scanning (CTCGAGCTCA) mutations in the tSL RNA gene was created between positions 42 and 129 (Fig. 1B) for transfection as stable episomes in L. tarentolae. Two mutations in the region from positions 42 to 49 were created: a mutant with alterations in bases 43 and 44 (43/44) was used to test a postulated SL RNA-U6 snRNA interaction (76), and a second mutant altered at positions 42 to 48 (42/48) changed all but the splice donor site with the equivalent linker-scanning 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 possible adjacent expression elements.
To examine the tSL RNA expression, total-cell RNA was separated on a medium-resolution gel system (Fig. 2). All tSL RNA genes were transcribed. RNA from mutants 42/48, 43/44, 52/59, 62/69, 100/109, 110/119, and 120/129 yielded the typical 96-nt tSL RNA. The 3'-most intron mutants, 70/79, 80/89, and 90/99, showed discrete increases in the tSL RNA length of less than 10 additional nucleotides, plus some faint higher-molecular-weight products that migrate above 215 nt. 100/109 showed, in addition to the 96-nt tSL RNA, a pattern of at least eight larger transcripts (~110 to 220 nt).
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Intron mutations affect 3'-end and cap 4 formation. Since gene-internal elements can affect the specificity of transcription initiation (27, 69), we tested the possibility that the changes in transcript size were due to incorrect transcription initiation. Primer extension analyses were performed with an oligonucleotide complementary to the exon tag sequence (Fig. 3). Extension products from accurately initiated and methylated tSL RNAs are expected to terminate predominantly 5 nt downstream of the true 5' end due to base methylation at the fourth position in the cap 4 structure (21, 55). Similar to WT SL RNA methylation in L. tarentolae (62), the control tSL RNA showed approximately 75% extension to position +5, as did the downstream mutations (110/119 and 120/129). Although all tSL RNAs showed primer extension products consistent with initiation at +1, only 42/48 and 43/44 conformed with the extension pattern expected for cap 4 methylation. 100/109 showed 50% methylation at position +4. The tSL RNAs mutated from 52/59 through 90/99 showed greatly reduced methylation (<5%) at +4 but appeared to retain base methylation of nucleotide +1, based on the predominant extension products at position +2.
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-32P-labeled 28/39-tag oligonucleotide. The
interpretation of the extension products is indicated in schematic form
to the right of the data, where cap 4 structures including the 7meG
cap, 2'-O-ribose methylations (m), and base methylations on
nt 1 (m6,6) and 4 (m,3) are indicated.
Sm-binding site and stem-loop III mutations result in extended 3' ends. To determine the precise sizes of the transcripts, the tSL RNAs were examined in single-nucleotide resolution gels (Fig. 4A). The tSL, 43/44, 100/109, 110/119, and 120/129 RNAs resolved predominantly as single bands. Doublet bands of equal intensity were observed for 42/48, 52/59, and 62/69 (see the shorter exposure of 62/69 in the right panel), in which the upper band corresponded to the band seen in tSL. The 70/79, 80/89, and 90/99 tSL RNAs were larger and comprised four major bands each. Based on the uniform 5' ends mapped by primer extension and on the ladder pattern of the single-nucleotide-resolution gel, the 3' ends of the 70/79, 80/89, and 90/99 transcripts terminated in a staggered fashion within the poly(T) tract, corresponding U residues 5 to 8.
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WT SL RNA poly(T)-tract termination. Because transcription initiation was accurate, the lengths of the tSL RNAs seen in 70/79, 80/89, and 90/99 suggested that transcription termination occurs in a staggered fashion in the poly(T) tract downstream of the SL RNA gene. Consistent with this proposal, transcription of the SL RNA does not proceed significantly beyond the poly(T) tract in nuclear run-on assays (40, 62). To determine if WT SL RNAs also terminate in the poly(T) tract, WT and 70/79 total RNA was resolved in a 6% acrylamide-urea gel (Fig. 4B). An overexposure of the WT lane showed the 96-nt SL RNA and a collection of larger bands that mirrored the tSL RNA accumulation pattern in 70/79, indicating that poly(T) tract termination products are produced during normal SL RNA synthesis.
These results suggest that formation of the SL RNA 3' end is a two-step process: transcription termination in the poly(T) tract followed by nucleolytic processing to/at the base of stem-loop III. Alternatively, two distinct termination signals may be operating in the WT SL RNA, one leading to the 96-nt transcript directly and the second resulting in the staggered termination in the poly(T) tract, or 3'-end formation may be a three-step process with transcription termination occurring downstream of the poly(T) tract followed by primary cleavage within the poly(U) tract and secondary cleavage at the mature 3' end.Transcription termination by the 3' poly(T) tract in vivo. The poly(T) tract location of 3' ends in some of the tSL RNAs, coupled with the increased length in the 100/109 mutant, suggested that the poly(T) tract itself was a termination element. Transcription termination mediated by poly(T) tracts is a hallmark of pol II (37) and pol III (8). Since the identity of the RNA polymerase that transcribes the SL RNA gene has been a subject of some contradictory data and since little is known about transcription termination in trypanosomatids, we performed a mutational analysis of the poly(T) tract.
Because 100/109 (2 T's) showed extended 3'-end products, the length of the poly(T) tract was altered in two nucleotide increments (Fig. 5A). These mutations were designed to define the number of T's necessary for termination. An 8-bp poly(T) tract terminated transcription efficiently, while 2-, 4-, and 6-bp poly(T) tracts accumulated higher-molecular-weight bands (Fig. 5B). The largest transcript (~220 nt) corresponds approximately to a 3' end within a pyrimidine-rich region in the pX sequence (TTTTGTTCCCTTT, at positions 201 to 213 relative to the start of transcription; positions 1415 to 1427 on the pX vector). The other products may correspond to termination by a low-processivity polymerase, to pause sites in an exonucleolytic pathway, or to secondary endonucleolytic cleavage sites exposed due to the transcription of the downstream sequences. The sizes of the high-molecular-weight products are not identical in all samples; this may be the result of secondary- or tertiary-structure effects on polymerase processivity or nucleolytic processing.
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Poly(T) tract termination with in vitro transcription
extracts.
We have demonstrated accurate transcription initiation
from the SL RNA gene by using in vitro transcription assays
(79) (Fig. 6A) similar to
those described for Trypanosoma and Leptomonas (26, 35); as in the related systems, cap 4 methylation is lacking in L. tarentolae extracts. Because in vitro products
could be detected by primer extension and blotting methods, we used this assay to monitor transcription termination. The in vitro products
from the constructs with altered poly(T) tracts (Fig. 5A) were compared
with their in vivo counterparts and analyzed by gel electrophoresis and
RNA hybridization (Fig. 6B) for transcription termination efficiency as
measured by an all-or-nothing appearance of bands in the in vivo
samples relative to the in vitro samples. We first noted that the in
vitro tSL-positive control did not comigrate with the in vivo tSL
product but migrated with transcripts that had staggered 3' ends in the
poly(T) tract. The differential migration cannot be ascribed to
variable 5'-end methylation, since the latter does not affect
electrophoretic mobility (71). We also noted three bands
near the top of the gel at 
300 nt in vitro that were not present in
the in vivo RNA samples. This transcript pattern was also seen for in
vitro products from 52/59 and 62/69 (data not shown), which resulted in
WT-sized transcripts in vivo.
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250 nt) than those seen in vivo (<215 nt). The constructs
with 8 and 10 T's (106/107 and tSL) led to poly(T)-tract-mediated
termination, as seen by the accumulation of poly(T)-tract products, but
was not as efficient as in vivo, since some higher-migrating bands were
also evident. The poly(T)-tract displacement mutation (100/109 + 150/159) showed primary termination products that corresponded to those
seen in vivo at around 155 nt, but none of the smaller accumulation
products, including no WT tSL RNA; small amounts of the 250-nt in
vitro-specific products were also visible. The 150/159 double
poly(T)-tract mutant accumulated products at both WT and the displaced
poly(T) tracts, confirming that in vitro termination was less efficient
than in vivo termination.
These results demonstrate the accurate and efficient termination in
nuclear extracts directed by poly(T) tracts of 8 nt. The lack of
WT-sized products in any of the in vitro products supports the
conclusion that primary transcript termination is mediated by the
poly(T) tract alone. In addition, the absence of minor products in the
range of 110 to 200 nt seen in the in vivo termination knockout mutants
(Fig. 5B) indicates that the presence of in vivo products is due to
processing and not to primary termination events. The combined results
of the in vivo and in vitro studies indicate that transcriptional
termination and 3' maturation are two distinct events. While it is
formally possible that the in vitro transcription system does not
recognize some alternative termination signal in the Sm-binding site or
stem-loop III region, it is unlikely based on additional mutagenesis
studies (see below).
The Sm-binding site affects 3'-end formation in vivo. Because 70/79 contained sequences affecting both 5'- and 3'-end formation and altered most of the Sm-binding site, we examined this sequence block in fine detail. A 2-bp transversion series was created from positions 70 through 81; in addition, 70/79 and 75/81 transversion mutations (TV) were made (Fig. 7A). Mutations 70/71 TV and 72/73 TV comprise part of stem-loop II (Fig. 1A).
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The structure of stem-loop III is necessary for proper 3'-end maturation. Mutations 80/89 and 90/99 disrupted stem-loop III and resulted in larger, undermethylated tSL RNAs (Fig. 3 and 4A). To examine further the effect of stem-loop III structure, a series of mutations that either disrupted or compensated 1 or 3 bp of the stem and that altered the loop sequence independent of the stem were created (Fig. 8). 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, while the triple-base mutations were designed to disrupt the stem thermodynamically.
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12 to 18 in the
presence (62, 66) and absence (66) of SL RNA gene
promoter activity prompted the suggestion that the artifacts were the
same and were a by-product of the runaround transcription in the
expression vector pX (62). However, when the IT is placed in
the stem-loop II structure (61) or removed altogether
(66), the 18 artifact does not appear in blotting
analyses. Thus, the placement of the IT in stem-loop III may be
responsible directly for the additional band seen in blotting analyses,
perhaps by interfering with 3'-end formation. This hypothesis was
tested by comparing IT-containing products generated in vivo to
products synthesized by the in vitro transcription system
(79) (Fig. 6). The in vitro products comigrated with the
18 product, and no mature-sized product was seen (Fig. 8C). Thus, we
conclude that the 18 artifact is due to the accumulation of poly(T)
tract termination products, consistent with its association with active
promoter elements and with impaired 3' processing due to the
introduction of an additional 44 nt to the loop of stem-loop III, and
does not correspond to the upstream primer extension products.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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We demonstrate that the SL RNA intron structure, content, and downstream poly(T) tract affect SL RNA termination and processing in an independent fashion. In L. tarentolae, SL RNA is most probably transcribed by pol II (62). Most aspects of pol II transcription in trypanosomatids are poorly understood, and little is known about how pol II transcription terminates in trypanosomatids (6, 39) and other organisms (64). However, termination of small RNA gene transcription is controlled by a well-defined 3' box (64). Transcription termination and 3'-end formation of the SL RNA can be separated into a two-step process: staggered transcription termination in the 3' poly(T) tract enhanced by the presence of stem-loop III, followed by nucleolytic processing to a defined 3' end at position 96 that is dependent on stem-loop III and the Sm-binding site.
Regarding the important question of the identity of the polymerase that
transcribes the SL RNA gene, it should be noted that relatively high
-amanitin resistance in pol II genes has been documented in
kinetoplastids (5, 24, 38, 41) as well as in other lower
eukaryotes (45, 57). The resulting differences in pol II and
pol III -amanitin sensitivities create difficulties in the
discrimination between these two polymerases on the basis of this
criterion, probably accounting for many of the conflicting conclusions
seen in the literature. Since the data from most inhibitors support pol
II transcription of the SL RNA gene in L. tarentolae (62), we will proceed with our discussion accordingly.
Termination and the poly(T) tract. Transcription termination by pol II has been studied primarily on protein-coding genes (74). The transcription termination signals are bipartite and include the polyadenylation signal (7) in conjunction with a variety of downstream elements, such as poly(T) tracts with intrinsic bent DNA but no adjacent upstream secondary structure (37), trans-acting factors binding to the DNA (4, 13, 14) or the RNA polymerase (75), or the structure of the transcript (56). Since long (>6-nt) poly(T) tracts may cause pausing but should not effect pol II termination on protein-coding genes, a different mechanism must exist for mRNA termination in trypanosomatids. Because transcription of kinetoplastid pre-mRNA is polycistronic and includes extensive polypyrimidine tracts as part of the trans-splicing signals, the factors involved in termination would have to be differentially recruited by the same polymerase. In this regard, it has been suggested that transcription driven by the T. brucei SL RNA gene promoter is terminated by the actin gene upstream polypyrimidine tract (49). In T. cruzi, transcription of an SL RNA-chloramphenicol acetyltransferase gene is driven by the SL RNA gene promoter, perhaps because the construct lacks the polypyrimidine splicing signal and has no poly(T) tracts longer than 5 nt in its coding region and 7 nt in the flanking sequences (46); it should also be noted that T. cruzi SL RNA genes have particularly lengthy poly(T) tracts, up to 31 nt (15), which may reflect a functional constraint for termination in this organism.
In the case of pol III, transcription terminates in a 4 T tract and is processed to the mature 3' end (8); termination is stimulated by La autoantigen (22, 23). In higher eukaryotes, termination-polyadenylation elements function only in the presence of the cognate snRNA-mRNA promoter (33, 34, 52). With the identification of the SL RNA gene termination element, a similar association between the SL RNA gene promoter and terminator elements can now be tested functionally in vivo and in vitro. The role of the poly(T) tract located at nt 98 to 107 as a transcription terminator is clear, since its disruption results in a variety of 3'-extended transcripts and since poly(T)-tract relocation 52 bp downstream generates the predicted ~155-nt products (Fig. 5). As a result of the disruption and relocation experiments, transcription proceeds into the flanking region, which has been shown in nuclear run-on assays to constitute a nontranscribed spacer between the tandemly arranged SL RNA genes (40, 62). The requirement of at least 6 T's for efficient transcriptional termination of the SL RNA gene in vivo and at least 8 T's in vitro argues against transcription by pol III, which terminates transcription at runs of 4 to 8 T's that have GC-rich flanking regions and lack flanking AA nucleotide pairs (8). Since tRNA and 5S genes in trypanosomatids generally follow these rules (see below), pol III might be expected to terminate prematurely at the 4 T's located in the Sm-binding site. In addition, by the same analogy, we would expect pol III to terminate transcription efficiently in the 4-T poly(T)-tract mutant (102/107), which lacks AA nucleotide pairs in the immediate flanking regions. Transcription termination at a poly(T) tract downstream of the SL RNA gene and the presence of an upstream modulating stem-loop structure are reminiscent of rho-independent termination in Escherichia coli and provide an alternative mechanism for pol II transcription termination for small-RNA genes in eukaryotes. Our model of pol II termination on the trypanosomatid SL RNA gene differs from that used for eukaryotic small-RNA genes that are transcribed by pol II and have downstream GTTN1-4AANARNAGA elements (32). Although present downstream of the pol II-transcribed SL RNA gene in Ascaris, the 3'-box sequence does not function as a terminator; termination signals lie within the transcribed region (28). A variety of poly(T) tracts are found downstream of the SL RNA genes in kinetoplastids. Within the leishmanias, L. donovani displays a range of 5 to 9 T's, L. major ranges from 8 to 9 T's, L. mexicana ranges from 8 to 10 T's, L. braziliensis ranges from 10 to 13 T's, and L. guyanensis, L. panamensis, and L. naiffi have interrupted T tracts T9CT10, T11CT6, and T8CT13, respectively (18). The trypanosomes display large homopolymeric poly(T) tracts ranging from 20 to 31 T's in T. cruzi (15, 19) and tracts interrupted by purine residues such as T5AT8AT7 in T. brucei (12). Purine interruptions are also seen in the nonpathogens Blastocrithidia (T10AT13) (17), Crithidia desouzai (T4GT3) (20), and Herpetomonas (T10AT8) (3). Because the possibility remains that the SL RNA gene is transcribed by pol III, we include a summary of putative pol III termination elements in L. tarentolae. Eighteen tRNA genes and two 5S rRNA genes have been characterized (48, 63, 68). The tRNA genes are presumed to be transcribed by pol III due to the presence of box A/box B or box A/box C motifs and downstream poly(T) tracts. Kinetoplastid tRNAs are typical in their transcription profiles with regard to the inhibitors-amanitin and Tagetitoxin (16, 62). Most of
the L. tarentolae tRNAs are encoded in clusters, and
transcription termination is likely to be effected by 4 to 6 T residues
present downstream of the transcribed region, although one exception is
found in relative isolation and has a putative termination signal of
T8AC7 (48). All the poly(T) tracts
lack adjacent AA dinucleotides, and the majority are flanked by GC-rich sequences. Among other genes in L. tarentolae presumed to be
transcribed by pol III promoters in associated tRNA genes
(51), U4 ends in a run of 4 T's (61), while
U-RNA B (the U3 homolog) has a stretch of 11 T's downstream of its
transcribed region (63) and U2 has a stretch of 9 T's
(78). The increased poly(T) tract length may be relevant in
the U-RNA B gene because the transcript itself has an internal stretch
of 4 T's. Functional assays will be necessary to determine if any of
these genes require more than 4 T's for pol III termination.
3'-end processing: Sm-binding site and stem-loop III. Disruption of the UUUUGG Sm-binding-site sequences or stem-loop III structure results in a loss of 3'-end processing in L. tarentolae (Fig. 7 and 8). Single-nucleotide disruptions of stem-loop III produce slightly modified 3' ends, but major disruptions of the stem portion of stem-loop III result in the accumulation of poly(T)-tract termination products ranging in size from 5 to 9 nt longer than the mature, WT transcript.
The presence of 3'-extended products in stably infected mutants (70/79, 80/89, and 90/99) suggests that transcription termination and 3'-end processing are separate events. This model is supported by the inability of an in vitro transcription extract to 3' process transcripts terminated in the poly(T) tract. The lack of 3' and 5' processing of tSL RNAs in the in vitro transcription nuclear extract suggests that both of these processing events occur in the cytoplasm of the cell rather than in the nuclear compartment. Consistent with this hypothesis and the nature of snRNP-processing pathways in other systems (36), fractionation studies indicate that the kinetoplastid SL RNA is present in its unspliced form in the cytoplasm as well as in the nucleus (31, 59). The lack of 5' methylation seen in our in vitro transcription system mirrors the results obtained in T. brucei (26). However, in other studies, nuclear extracts were found to perform as well as whole-cell extracts did for the 5' methylation of in vitro-transcribed SL RNA (72). Future studies will explore the ability of Leishmania cytoplasmic, nuclear, or whole-cell extracts to process poly(T)-tract-terminated tSL RNA substrates and to identify the nuclease responsible. 3' processing is a critical step for SL RNA maturation, since unprocessed tSL RNAs are unable to participate in the trans-splicing reaction (65). The mode of 3' processing to the base of stem-loop III remains to be determined. It is likely to be an enzymatic reaction that could include endonucleolytic attack at the base of secondary structures (for example, RNase P processing of the 5' ends of tRNAs of Escherichia coli) or exonucleolytic trimming (for example, exonucleolytic trimming of U6 [9]). In this regard, we note that the related kinetoplastid T. cruzi possesses an exonuclease activity that removes uridine but stops at adenosine (81), which is a candidate for SL RNA 3' processing. Given the general lack of knowledge about transcription in trypanosomes, our work on the promoter (80) and transcription termination elements is the first step toward a global understanding of pol II function in these primitive eukaryotes. The definition of these elements can now be exploited for continued study of the SL RNA itself, which is so central to kinetoplastid pre-mRNA processing. In addition, these elements can be used to drive the expression of other RNAs, natural or engineered, that will expedite the study of other genes from a functional rather than transcriptional point of view.| |
ACKNOWLEDGMENTS |
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We thank Steve Beverley for the pX plasmid, Arthur Günzl for discussion of the in vitro transcription system and its potential for termination, T. Guy Roberts for stimulating discussions, and Doug Black, Larry Feldman, and Dan Ray for critical reading of the manuscript.
This work was supported by a National Institutes of Health grant (AI34536). N.R.S. is a postdoctoral trainee on Microbial Pathogenesis Training Grant 2-T32-AI-07323.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095. Phone: (310) 825-4195. Fax: (310) 206-3865. E-mail: dc{at}ucla.edu.
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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| 1. | Agabian, N. 1990. Trans splicing of nuclear pre-mRNAs. Cell 61:1157-1160[Medline]. |
| 2. |
Agami, R.,
R. Aly,
S. Halman, and M. Shapira.
1994.
Functional analysis of cis-acting DNA elements required for expression of the SL RNA gene in the parasitic protozoan Leishmania amazonensis.
Nucleic Acids Res.
22:1959-1965 |
| 3. |
Aksoy, S.
1992.
Spliced leader RNA and 5S rRNA genes in Herpetomonas spp. are genetically linked.
Nucleic Acids Res.
20:913 |
| 4. | Ashfield, R., A. J. Patel, S. A. Bossone, H. Brown, R. D. Campbell, K. B. Marcu, and N. J. Proudfoot. 1994. MAZ-dependent termination between closely spaced human complement genes. EMBO J. 13:5656-5667[Medline]. |
| 5. |
Ben Amar, M. F.,
A. Pays,
P. Tebabi,
B. Dero,
T. Seebeck,
M. Steinert, and E. Pays.
1988.
Structure and transcription of the actin gene of Trypanosoma brucei.
Mol. Cell. Biol.
8:2166-2176 |
| 6. | Berberof, M., A. Pays, S. Lips, P. Tebabi, and E. Pays. 1996. Characterization of a transcription terminator of the procyclin PARP A unit of Trypanosoma brucei. Mol. Cell. Biol. 16:914-924[Abstract]. |
| 7. | Birse, C. E., B. A. Lee, K. Hansen, and N. J. Proudfoot. 1997. Transcriptional termination signals for RNA polymerase II in fission yeast. EMBO J. 16:3633-3643[Medline]. |
| 8. | Bogenhagen, D. F., and D. D. Brown. 1981. Nucleotide sequences in Xenopus 5S DNA required for transcription termination. Cell 24:261-270[Medline]. |
| 9. |
Booth, B. L. J., and B. F. Pugh.
1997.
Identification and characterization of a nuclease specific for the 3' end of the U6 small nuclear RNA.
J. Biol. Chem.
272:984-991 |
| 10. | Boothroyd, J. C., D. A. Campbell, and R. E. Sutton. 1985. Expression of surface antigen genes in Trypanosoma brucei involves a novel system of discontinuous transcription, p. 61-66. In R. A. Lerner, R. M. Channock, and F. Brown (ed.), Vaccines 85: modern approaches to vaccines. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 11. | Bruzik, J. P., K. Van Doren, D. Hirsh, and J. A. Steitz. 1988. Trans splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature 335:559-562[Medline]. |
| 12. | Campbell, D. A., D. A. Thornton, and J. C. Boothroyd. 1984. Apparent discontinuous transcription of Trypanosoma brucei variant surface antigen genes. Nature 311:350-355[Medline]. |
| 13. | Connelly, S., and J. L. Manley. 1989. A CCAAT box sequence in the adenovirus major late promoter functions as part of an RNA polymerase II termination signal. Cell 57:561-571[Medline]. |
| 14. |
Connelly, S., and J. L. Manley.
1989.
RNA polymerase II transcriptional termination is mediated specifically by protein binding to a CCAAT box sequence.
Mol. Cell. Biol.
9:5254-5259 |
| 15. |
De Lange, T.,
T. M. Berkvens,
H. J. Veerman,
A. C. Frasch,
J. D. Barry, and P. Borst.
1984.
Comparison of the genes coding for the common 5' terminal sequence of messenger RNAs in three trypanosome species.
Nucleic Acids Res.
12:4431-4443 |
| 16. |
Fantoni, A.,
A. O. Dare, and C. Tschudi.
1994.
RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.
Mol. Cell. Biol.
14:2021-2028 |
| 17. | Fernandes, O., and D. A. Campbell. Unpublished results. |
| 18. | Fernandes, O., V. K. Murthy, U. Kurath, W. M. Degrave, and D. A. Campbell. 1994. Mini-exon gene variation in human pathogenic Leishmania species. Mol. Biochem. Parasitol. 66:261-271[Medline]. |
| 19. | Fernandes, O., N. R. Sturm, R. Derré, and D. A. Campbell. 1998. The mini-exon gene: a genetic marker for zymodeme III of Trypanosoma cruzi. Mol. Biochem. Parasitol. 95:129-133[Medline]. |
| 20. | Fernandes, O., M. M. G. Teixeira, N. R. Sturm, M. A. Sousa, E. P. Camargo, W. M. Degrave, and D. A. Campbell. 1997. Mini-exon gene sequences define six groups within the genus Crithidia. J. Eukaryot. Microbiol. 44:535-539[Medline]. |
| 21. |
Freistadt, M. S.,
G. A. M. Cross, and H. D. Robertson.
1988.
Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A.
J. Biol. Chem.
263:15071-15075 |
| 22. | Gottlieb, E., and J. A. Steitz. 1989. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J. 8:851-861[Medline]. |
| 23. | Gottlieb, E., and J. A. Steitz. 1989. The RNA binding protein La influences both the accuracy and the efficiency of RNA polymerase III transcription in vitro. EMBO J. 8:841-850[Medline]. |
| 24. | Grondal, E. J., R. Evers, K. Kosubek, and A. W. C. A. Cornelissen. 1989. Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III. EMBO J. 8:3383-3389[Medline]. |
| 25. |
Günzl, A.,
C. Tschudi,
V. Nakaar, and E. Ullu.
1995.
Accurate transcription of the Trypanosoma brucei U2 small nuclear RNA gene in a homologous extract.
J. Biol. Chem.
270:17287-17291 |
| 26. | Günzl, A., E. Ullu, M. Dorner, S. P. Fragoso, K. F. Hoffmann, J. D. Milner, Y. Morita, E. K. Nguu, S. Vanacova, S. Wunsch, A. O. Dare, H. Kwon, and C. Tschudi. 1997. Transcription of the Trypanosoma brucei spliced leader RNA gene is dependent only on the presence of upstream regulatory elements. Mol. Biochem. Parasitol. 85:67-76[Medline]. |
| 27. |
Hannon, G. E.,
G. J. Hannon,
P. A. Maroney, and T. W. Nilsen.
1994.
Transcription of a nematode U1 small nuclear RNA in vitro. 3'-end formation requires cis-acting elements within the coding sequence.
J. Biol. Chem.
269:12387-12390 |
| 28. | Hannon, G. J., P. A. Maroney, D. G. Ayers, J. D. Shambaugh, and T. W. Nilsen. 1990. Transcription of a nematode trans-spliced leader RNA requires internal elements for both initiation and 3' end-formation. EMBO J. 9:1915-1921[Medline]. |
| 29. | Harris, K. A., Jr., D. M. Crothers, and E. Ullu. 1995. In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations. RNA 1:351-362[Abstract]. |
| 30. | Hartree, D., and V. Bellofatto. 1995. Essential components of the mini-exon gene promoter in the trypanosomatid Leptomonas seymouri. Mol. Biochem. Parasitol. 71:27-39[Medline]. |
| 31. |
Hartshorne, T., and N. Agabian.
1993.
RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.
Mol. Cell. Biol.
13:144-154 |
| 32. | Hernandez, N. 1992. Transcription of vertebrate snRNA genes and related genes, p. 281-313. In K. Yamamoto, and S. L. McKnight (ed.), Transcriptional regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 33. | Hernandez, N., and R. Lucito. 1988. Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation. EMBO J. 7:3125-3134[Medline]. |
| 34. | Hernandez, N., and A. M. Weiner. 1986. Formation of the 3' end of U1 snRNA requires compatible snRNA promoter elements. Cell 47:249-258[Medline]. |
| 35. | Huie, J. L., P. He, and V. Bellofatto. 1997. In vitro transcription of the Leptomonas seymouri SL RNA and U2 snRNA genes using homologous cell extracts. Mol. Biochem. Parasitol. 90:183-192[Medline]. |
| 36. | Izaurralde, E., and I. W. Mattaj. 1992. Transport of RNA between nucleus and cytoplasm. Semin. Cell Biol. 3:279-288[Medline]. |
| 37. | Kerppola, T. K., and C. M. Kane. 1990. Analysis of the signals for transcription termination by purified RNA polymerase II. Biochemistry 29:269-278[Medline]. |
| 38. | Köck, J., and A. W. C. A. Cornelissen. 1991. Characterization of the RNA polymerases of Crithidia fasciculata. Mol. Microbiol. 5:835-842[Medline]. |
| 39. |
König, E.,
H. Delius,
M. Carrington,
R. O. Williams, and I. Roditi.
1989.
Duplication and transcription of procyclin genes in Trypanosoma brucei.
Nucleic Acids Res.
17:8727-8739 |
| 40. | Kooter, J. M., T. De Lange, and P. Borst. 1984. Discontinuous synthesis of mRNA in trypanosomes. EMBO J. 3:2387-2392[Medline]. |
| 41. |
Laird, P. W.,
J. M. Kooter,
N. Loosbroek, and P. Borst.
1985.
Mature mRNAs of Trypanosoma brucei possess a 5' cap acquired by discontinuous RNA synthesis.
Nucleic Acids Res.
13:4253-4266 |
| 42. | Laird, P. W., J. C. Zomerdijk, D. de Korte, and P. Borst. 1987. In vivo labelling of intermediates in the discontinuous synthesis of mRNAs in Trypanosoma brucei. EMBO J. 6:1055-1062[Medline]. |
| 43. |
LeBowitz, J. H.,
C. M. Coburn,
D. McMahon-Pratt, and S. M. Beverley.
1990.
Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.
Proc. Natl. Acad. Sci. USA
87:9736-9740 |
| 44. | LeCuyer, K. A., and D. M. Crothers. 1993. The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms. Biochemistry 32:5301-5311[Medline]. |
| 45. |
Lioutas, C., and E. Tannich.
1995.
Transcription of protein-coding genes in Entamoeba histolytica is insensitive to high concentrations of -amanitin.
Mol. Biochem. Parasitol.
73:259-261[Medline].
|
| 46. | Lu, H.-Y., and G. A. Buck. 1991. Expression of an exogenous gene in Trypanosoma cruzi epimastigotes. Mol. Biochem. Parasitol. 44:109-114[Medline]. |
| 47. | Lücke, S., G. L. Xu, Z. Palfi, M. Cross, V. Bellofatto, and A. Bindereif. 1996. Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap 4 formation. EMBO J. 15:4380-4391[Medline]. |
| 48. | Lye, L.-F., D.-H. T. Chen, and Y. Suyama. 1993. Selective import of nuclear-encoded tRNAs into mitochondria of the protozoan Leishmania tarentolae. Mol. Biochem. Parasitol. 58:233-246[Medline]. |
| 49. | McAndrew, M., S. Graham, C. Hartmann, and C. Clayton. 1998. Testing promoter activity in the trypanosome genome: Isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription. Exp. Parasitol. 90:65-76[Medline]. |
| 50. |
Mottram, J.,
K. L. Perry,
P. M. Lizardi,
R. Luhrmann,
N. Agabian, and R. G. Nelson.
1989.
Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.
Mol. Cell. Biol.
9:1212-1223 |
| 51. | Nakaar, V., C. Tschudi, and E. Ullu. 1995. An unusual liaison: small nuclear and cytoplasmic RNA genes team up with tRNA genes in trypanosomatid protozoa. Parasitol. Today 11:225-228. |
| 52. | Neuman de Vegvar, H. E., E. Lund, and J. E. Dahlberg. 1986. 3' end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters. Cell 47:259-266[Medline]. |
| 53. |
Nilsen, T. W.,
J. Shambaugh,
J. Denker,
G. Chubb,
C. Faser,
L. Putnam, and K. Bennett.
1989.
Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.
Mol. Cell. Biol.
9:3543-3547 |
| 54. | Nunes, L. R., M. R. Carvalho, A. M. Shakarian, and G. A. Buck. 1997. The transcription promoter of the spliced leader gene from Trypanosoma cruzi. Gene 188:157-168[Medline]. |
| 55. |
Perry, K. L.,
K. P. Watkins, and N. Agabian.
1987.
Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader.
Proc. Natl. Acad. Sci. USA
84:8190-8194 |
| 56. |
Pribyl, T. M., and H. G. Martinson.
1988.
Transcriptional termination at the chicken  H-globin gene.
Mol. Cell. Biol.
8:5369-5377 |
| 57. |
Quon, D. V. K.,
M. G. Delgadillo, and P. J. Johnson.
1996.
Transcription in the early diverging eukaryote Trichomonas vaginalis: an unusual RNA polymerase II and -amanitin-resistant transcription of protein-coding genes.
J. Mol. Biol.
43:253-262.
|
| 58. | Roberts, T. G., J. M. Dungan, K. P. Watkins, and N. Agabian. 1996. The SLA RNA gene of Trypanosoma brucei is organized in a tandem array which encodes several small RNAs. Mol. Biochem. Parasitol. 83:163-174[Medline]. |
| 59. |
Roberts, T. G.,
N. R. Sturm,
B. K. Yee,
M. C. Yu,
T. Hartshorne,
N. Agabian, and D. A. Campbell.
1998.
Three small nucleolar RNAs identified from the spliced leader-associated RNA locus in kinetoplastid protozoans.
Mol. Cell. Biol.
18:4409-4417 |
| 60. |
Rudenko, G.,
M. G.-S. Lee, and L. H. T. Van der Ploeg.
1992.
The PARP and VSG genes of Trypanosoma brucei do not resemble RNA polymerase II transcription units in sensitivity to Sarkosyl in nuclear run-on assays.
Nucleic Acids Res.
20:303-306 |
| 61. | Saito, R. M., and D. A. Campbell. Unpublished results. |
| 62. | Saito, R. M., M. G. Elgort, and D. A. Campbell. 1994. A conserved upstream element is essential for transcription of the Leishmania tarentolae mini-exon gene. EMBO J. 13:5460-5469[Medline]. |
| 63. | Shi, X., D.-H. T. Chen, and Y. Suyama. 1994. A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria. Mol. Biochem. Parasitol. 65:23-37[Medline]. |
| 64. | Shilatifard, A., J. W. Conaway, and R. C. Conaway. 1997. Mechanism and regulation of transcriptional elongation and termination by RNA polymerase II. Curr. Opin. Genet. Dev. 7:199-204[Medline]. |
| 65. | Sturm, N. R., and D. A. Campbell. Unpublished results. |
| 66. |
Sturm, N. R.,
J. Fleischmann, and D. A. Campbell.
1998.
Efficient trans-splicing of mutated spliced leader exons in Leishmania tarentolae.
J. Biol. Chem.
273:18689-18692 |
| 67. |
Sturm, N. R.,
R. Kuras,
S. Büschlen,
W. Sakamoto,
K. L. Kindle,
D. B. Stern, and F.-A. Wollman.
1994.
The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts.
Mol. Cell. Biol.
14:6171-6179 |
| 68. | Suyama, Y., S. Wong, and D. A. Campbell. 1998. Regulated tRNA import in Leishmania mitochondria. Biochim. Biophys. Acta 1396:138-142[Medline]. |
| 69. | Tebb, G., and I. W. Mattaj. 1988. Positionally exact initiation is required for the formation of a stable RNA polymerase II transcription complex in vivo. EMBO J. 7:3785-3792[Medline]. |
| 70. |
Ullu, E., and C. Tschudi.
1990.
Permeable trypanosome cells as a model system for transcription and trans-splicing.
Nucleic Acids Res.
18:3319-3326 |
| 71. |
Ullu, E., and C. Tschudi.
1991.
Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.
Proc. Natl. Acad. Sci. USA
88:10074-10078 |
| 72. |
Ullu, E., and C. Tschudi.
1995.
Accurate modification of the trypanosome spliced leader cap structure in a homologous cell-free system.
J. Biol. Chem.
270:20365-20369 |
| 73. | Ullu, E., C. Tschudi, and A. Günzl. 1996. Trans-splicing in trypanosomatid protozoa, p. 115-133. In D. F. Smith, and M. Parsons (ed.), Molecular biology of parasitic protozoa. IRL Press, Oxford, United Kingdom. |
| 74. | Uptain, S. M., C. M. Kane, and M. J. Chamberlin. 1997. Basic mechanisms of transcript elongation and its regulation. Annu. Rev. Biochem. 66:117-172[Medline]. |
| 75. |
Xie, Z., and D. H. Price.
1996.
Purification of an RNA polymerase II transcript release factor from Drosophila.
J. Biol. Chem.
271:11043-11046 |
| 76. |
Xu, G. L.,
B. Wieland, and A. Bindereif.
1994.
Trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.
Mol. Cell. Biol.
14:4565-4570 |
| 77. |
Xu, Y.,
H. Ben-Shlomo, and S. Michaeli.
1997.
The U5 RNA of trypanosomes deviates from the canonical U5 RNA: the Leptomonas collosoma U5 RNA and its coding gene.
Proc. Natl. Acad. Sci. USA
94:8473-8478 |
| 78. | Yee, B. K., and D. A. Campbell. Unpublished results. |
| 79. | Yu, M. C., T. G. Roberts, and D. A. Campbell. Unpublished results. |
| 80. | Yu, M. C., N. R. Sturm, R. M. Saito, T. G. Roberts, and D. A. Campbell. 1998. Single nucleotide resolution of promoter activity and protein binding for the Leishmania tarentolae spliced leader RNA gene. Mol. Biochem. Parasitol. 94:265-281[Medline]. |
| 81. |
Zwierzynski, T. A.,
G. Widmer, and G. A. Buck.
1989.
In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi.
Nucleic Acids Res.
17:4647-4660 |
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