Tb927.10.6900 encodes the glucosyltransferase involved in synthesis of base J in Trypanosoma brucei.
ABSTRACT: Base J is a DNA modification found in the genome of Trypanosoma brucei and all other kinetoplastids analyzed, where it replaces a small fraction of Ts, mainly in telomeric and chromosome-internal transcription initiation and termination regions. The synthesis of base J is a two-step process whereby a specific T is converted to HOMedU (hydroxymethyldeoxyuridine) and subsequently glucosylated to generate J. The thymidine hydroxylases (JPB1 and JBP2) that catalyze the first step have been characterized, but the identity of the glucosyltransferase catalyzing the second step has proven elusive. Recent bioinformatic analysis by Iyer et al. (Nucleic Acids Res 2013;41:7635) suggested that Tb927.10.6900 encodes the glucosyltransferase (HmdUGT) responsible for converting HOMedU to J in T. brucei. We now present experimental evidence to validate this hypothesis; null mutants of Tb927.10.6900 are unable to synthesize base J. Orthologues from related kinetoplastids show only modest conservation, with several insertion sequences found in those from Leishmania and related genera.
Project description:Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA base (?-d-glucosyl-hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTUs) in T. cruzi. The loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate base J synthesis (JBP1 and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression. The affected genes include virulence genes, and the resulting parasites are defective in host cell invasion and egress. These studies indicate that base J is an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides the first biological role of the only hypermodified DNA base in eukaryotes.
Project description:Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (?-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.
Project description:Base J is a hypermodified DNA base localized primarily to telomeric regions of the genome of Trypanosoma brucei. We have previously characterized two thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 is a chromatin re-modeling protein that induces de novo J-synthesis, allowing JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and high-throughput sequencing. This genome-wide analysis revealed an enrichment of base J at regions flanking polymerase II polycistronic transcription units (Pol II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the telomeric regions. However, the maintenance of J at JBP1-specific regions is dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania major also contain base J highlights the functional importance of the modified base at Pol II PTUs within members of the kinetoplastid family. The regulation of J synthesis/localization by two THs and potential biological function of J in regulating kinetoplastid gene expression is discussed.
Project description:Iridoids are one of the most widely distributed secondary metabolites in higher plants. They are pharmacologically active principles in various medicinal plants and key intermediates in the biosynthesis of monoterpenoid indole alkaloids as well as quinoline alkaloids. Although most iridoids are present as 1-O-glucosides, the glucosylation step in the biosynthetic pathway has remained obscure. We isolated a cDNA coding for UDP-glucose:iridoid glucosyltransferase (UGT85A24) from Gardenia jasminoides. UGT85A24 preferentially glucosylated the 1-O-hydroxyl group of 7-deoxyloganetin and genipin but exhibited only weak activity toward loganetin and no activity toward 7-deoxyloganetic acid. This suggests that, in the biosynthetic pathway of geniposide, a major iridoid compound in G. jasminoides, glucosylation occurs after methylation of 7-deoxyloganetic acid. UGT85A24 showed negligible activity toward any acceptor substrates other than iridoid aglycones. Thus, UGT85A24 has a remarkable specificity for iridoid aglycones. The mRNA level of UGT85A24 overlaps with the marked increase in genipin glucosylation activity in the methyl jasmonate-treated cell cultures of G. jasminoides and is related to iridoid accumulation in G. jasminoides fruits.
Project description:?-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (?-D-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gHmdC), for the accurate detection of ?-D-glucosyl-5-hydroxymethyl-2'-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2'-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.?
Project description:In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37 degrees C, it is essential for parasite growth and survival at 40 degrees C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.
Project description:Unlike other eukaryotes, the protein coding genes of Trypanosoma cruzi are arranged into large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, trypanosomes rely on post-transcriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA base (-D-glucosyl hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTU) in T. cruzi. loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate J synthesis (JBP1 and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression. genes include virulence genes and the resulting parasites are defective in host cell invasion and egress. These studies indicate that base J represents an epigenetic factor regulating Pol II transcription initiation in kinetoplastids, and provides the first biological role of the only hyper-modified DNA base in eukaryotes. This entry contains the results using SLT, digital gene expression profiling of tags read off the spliced leader sequence of all mature trypanosomatid messages, for the life-cycle of T. cruzi, four stages, in wild-type as well as JBP1 KO cells. Overall design: This entry contains the results using SLT, digital gene expression profiling of tags read off the spliced leader sequence of all mature trypanosomatid messages, for the life-cycle of T. cruzi, four stages, in wild-type as well as JBP1 KO cells, for eight libraries in total.
Project description:Base J, ?-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.
Project description:Anthraquinones are widely distributed in various organisms and known as bioactive ingredients. Some of the anthraquinones accumulate as glycosides in higher plants. Plant secondary product glycosyltransferases (PSPGs) are the well-characterized enzymes producing plant secondary metabolite glycosides. However, PSPGs involved in the formation of anthraquinone glycosides remains unclear. The rhizome of Rheum palmatum contains anthraquinones as laxative agents, some of which are accumulated as glucosides. We isolated a glucosyltransferase, R. palmatum UDP-glycosyltransferase (RpUGT) 1 from the rhizome of R. palmatum, and characterized functionally. RpUGT1 glucosylated emodin yielding emodin-6-O-glucoside, and it also glucosylated rhapontigenin, a compound belonging to stilbenes, yielding rhaponticin. The expression patterns of RpUGT1 and the accumulation of the metabolites revealed that RpUGT1 contributes to the production of these glucosides in R. palmatum. These results may provide important information for the substrate recognition of the PSPGs for anthraquinones and stilbenes.
Project description:Base J or beta-d-glucosylhydroxymethyluracil is a DNA modification replacing a fraction of thymine in the nuclear DNA of kinetoplastid parasites and of Euglena. J is located in the telomeric sequences of Trypanosoma brucei and in other simple repeat DNA sequences. In addition, J was found in the inactive variant surface glycoprotein (VSG) expression sites, but not in the active expression site of T. brucei, suggesting that J could play a role in transcription silencing in T. brucei. We have now looked at the distribution of J in the genomes of other kinetoplastid parasites. First, we analyzed the DNA sequences immunoprecipitated with a J-antiserum in Leishmania major Friedlin. Second, we investigated the co-migration of J- and telomeric repeat-containing DNA sequences of various kinetoplastids using J-immunoblots and Southern blots of fragmented DNA. We find only approximately 1% of J outside the telomeric repeat sequences of Leishmania sp. and Crithidia fasciculata, in contrast to the substantial fraction of non-telomeric J found in T. brucei, Trypanosoma equiperdum and Trypanoplasma borreli. Our results suggest that J is a telomeric base modification, recruited for other (unknown) functions in some kinetoplastids and Euglena.