Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10 -7 per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.

We have developed a procedure for the affinity purification of small nuclear ribonucleoproteins (snRNPs) of Trypanosoma brucei (U2 and U4/U6 snRNPs), which are essential for trans splicing. Each of these snRNPs can be specifically and efficiently selected from T. brucei extracts through biotinylated antisense 2'-0-methylated RNA oligonucleotides immobilized on streptavidin-agarose. Protein analysis revealed a set of five low molecular weight polypeptides common to the U2 and U4/U6 snRNPs and the spliced leader RNP. In addition, several U2 and U4/U6 snRNP-specific protein components were identified. Using monoclonal antibodies against human snRNP proteins, we could not detect any significant cross-reaction with the trypanosomal U2 snRNP proteins. Thus, the trypanosomal snRNPs exhibit principal differences from the higher eukaryotic snRNPs not only in their RNA but also in their protein components.trans splicing in trypanosomatids and nematodes follows a two-step mechanism of cleavage-ligation reactions analogous to nuclear pre-mRNA splicing (cis splicing) of other eukaryotes. In trypanosomes, a mini-exon derived from the 5' end of the spliced leader (SL) RNA is trans-spliced onto every protein-coding exon of long, polycistronic pre-mRNAs (reviewed in refs. 1 and 2). As in cis splicing, small nuclear RNAs (snRNAs) function as essential splicing factors in trans splicing (3): trypanosomal U2, U4, and U6 RNAs have been identified (4-6) that are in the form of U2, U4/U6, and U6 small nuclear ribonucleoproteins (snRNPs) (7, 27); however, no U5 RNA homologue is known in trypanosomes. The SL RNP can be considered a chimeric molecule that possesses characteristics of an snRNP and carries the mini-exon sequence at the SL RNA 5' terminus, thereby possibly performing the role of the U1 snRNP (8-10). In contrast to the mammalian snRNPs (reviewed in ref. 11), we know little about the RNA-protein structure of the trypanosomal snRNPs and their mechanism of action during trans splicing. The Trypanosoma brucei snRNAs deviate in many aspects from their strongly conserved eukaryotic counterparts (reviewed in ref.2). Thus, through the characterization and comparison of cis-and trans-spliceosomal snRNPs, we expect to gain more information about the specific requirements of trans splicing. We have developed procedures for the affinity purification of SL, U2, and U4/U6 snRNPs on the basis of biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotides, resulting in the identification of a set of five common proteins and additional, snRNP-specific proteins. Interestingly, no immunological relationship of these proteins with mammalian snRNP proteins could be detected.

β-d-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. InTrypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of theT. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

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trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8,14, 12.5, and 10 kDa) (36). In contrast, no Ul and U5 RNA homologs are known in trypanosomes; the SL RNA, however, has been proposed to function both as a Ul-like snRNP and as a donor of its own 5'-terminal 40 nucleotides (11,54). This has recently been directly demonstrated in a mammalian in vitro system, in which the 5'-terminal domain of Leptomonas collosoma SL RNA could substitute for the 5' end of Ul snRNA in pre-mRNA cis splicing (10). In support of such a functional role, the SL sequence as well as the overall secondary structure of the SL RNA are strongly conserved between different trypanosome and nematode species (11,22,32,40; for the T. brucei SL RNA structure, see Fig. 3A).The trypanosomal snRNAs deviate most significantly from the otherwise strongly conserved sequence and secondary structure of eucaryotic snRNAs (18). For example, none of the known T. brucei snRNAs carry an Sm binding site, a sequence motif with the consensus PuAU3 4NUGPu, which is present in Ul, U2, U4, and U5 RNAs of all other eucaryotic species and usually located in a single-stranded region between two stem-loops (18, 28). In the mammalian system, the Sm binding site is essential for the trimethyl capping of snRNAs and for the cytoplasmic-nuclear transport of snRNPs (reviewed in reference 31). Furthermore, human anti-Sm antibodies do not cross-react with T. brucei snRNPs (33). There are other deviations of the T. brucei snRNAs from the eucaryotic consensus sequences and secondary structures, such as the lack of stem-loop III and of a branch point interaction region in T. brucei U2 RNA (20,36,53). Finally, the cap structure of the SL RNA is exceptional 5516 MOLECULAR AND CELLULAR BIOLOGY, Nov. 1991, p. 5516-5526

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5′‐terminal mini‐exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini‐exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem‐loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini‐exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini‐exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA‐RNA interactions in the trans spliceosome.

SummaryThe nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base b b b b -D -glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J-binding protein (JBP) in T. brucei . The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.