Leishmania parasites infect humans worldwide, leading to substantial morbidity and mortality. Transcription of protein-coding genes in Leishmania and other trypanosomatids initiates at the 5'end of a small number of large polycistronic transcription units (PTUs), and mature mRNAs are generated by trans-splicing of the primary transcript. However, little is known about how transcription is terminated in Leishmania. Base J is a hyper-modified DNA base that replaces ~1% of T in the nuclear DNA in Leishmania and related organisms. When one of the genes (JBP2) involved in J biosynthesis is disrupted in L. tarentolae promastigotes, they lose ~70% of their J, leading to massive transcriptional read-through at the convergent strand-switch regions between PTUs (and an increase in false-starts where transcription initiates). Furthermore, JPB2 dKO parasites become hypersensitive to growth in bromodeoxyuridine (BrdU), leading to a further reduction in J, even more read-through, and cell death. We postulate that iJ serves as a critical signal for transcription termination, and that loss of proper termination in J-deficient cells kills the paraste. In this project, we will critically assess this hypothesis by using a combination of state-of-the-at approaches to further elucidate J biosynthesis and function in Leishmania.
In Aim 1, we will use RNA-seq to analyze samples isolated at various times after disruption of the JBP2 and JBP1 genes, as well as BrdU and DMOG treatment, to identify mRNAs whose levels increase or decrease after iJ loss (with potentially lethal consequences), as well compensatory changes in gene expression that enable L. tarentolae to cope with this insult.
Aim 2 will utilize single-molecule real-time (SMRT) sequencing to identify the exact T residues that are converted to J, and will use systematic modification of these sequences to identify the minimal information required for de novo J synthesis, as well as for J "spreading".
In Aim 3, we will use in vivo and i vitro transcription assays, genome-wide chromatin mapping, as well as fluorescence in situ hybridization and chromosome conformation capture experiments to determine whether J terminates transcription either by directly blocking the RNA polymerase, by inducing chromatin changes, or by recruitment of chromatin to specific sub-nuclear "silencing domains".
Aim 4 will use DNA affinity and PTP-tagged protein affinity approaches, coupled with quantitative mass spectrometry-based proteomics, to identify proteins associated with iJ insertion and those recruited by iJ at transcription termination sites. The results from these experiments will be combined to build a more complete model of the molecular processes associated with J-mediated transcription termination. The proposed studies will increase our understanding of the role of J in controlling transcription in these organisms, and may provide insight into a possible role for similar base modifications in other eukaryotes. Furthermore, since base J is not found in the mammalian hosts of Leishmania (including humans), this information will provide a new opportunity for the development of novel chemotherapeutic agents against these important parasites.
Human infections with the various species of Leishmania parasite cause substantial morbidity and mortality in tropical and sub-tropical countries world-wide. These organisms appear to differ from their animal hosts in the way that they transcribe the genetic information in their genomic DNA into messenger RNAs, which are used to make the proteins. These studies are designed to elucidate parasite-specific differences in this vital process, which we hope may be exploited for the development of novel anti-Leishmania drugs.
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