Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei.
Bottom Line: Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination.Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death.In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes.
Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Davison Life Sciences Building, 120 Green Street, Athens, GA 30602-7229, USA.Show MeSH
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Mentions: Loss of base J results in readthrough transcription and the production of antisense RNAs in L. major. (A) Anti-base J dot blot analysis of WT L. major. A 2-fold serial dilution of genomic DNA was spotted onto a membrane and incubated with anti-base J antisera. −: DMSO; +: 5-mM DMOG. The membrane was stripped and probed using a radio-labeled beta tubulin probe as a loading control. (B) Anti-base J IP qPCR analysis. The average of three independent IPs is plotted as the percent IP relative to the total input material. All IPs were background subtracted using a no antibody control. White bars: DMSO; black bars: DMOG. Seven cSSRs enriched for base J are shown and one previously identified J negative cSSR (28.2) as a negative control (see Supplementary Table S1 for genomic location). Error bars represent the standard deviation. (C) Upper panels, small RNA sequencing reads for three cSSRs where J loss led to readthrough transcription are shown. Small RNA reads are plotted as reads per million reads mapped (rpm). Upper graphs: DMSO; lower graphs: DMOG. ORFs and the genomic location (kb) are shown above the graphs. Blue: top strand; red: bottom strand. Lower panels: cSSR 35.6 illustrates a region where J was not reduced by DMOG (see Figure 1B) and there was no readthrough defect; cSSR 36.3 shows a site containing tRNA genes on both DNA strands where J was reduced by DMOG (see Supplementary Figure S1C), but did not result in a readthrough defect; and HT site 20.3 shows a non-cSSR termination site where J loss (see Supplementary Figure S1C) resulted in a termination defect. (D) A metaplot summarizing the readthrough defect at cSSRs (n = 36, 3 discarded) aligned by their TTS, shown as position 0 on the x-axis. Meta coverage of each sample was normalized by the mean meta coverage of upstream of TTS (see the Materials and Methods section).
Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Davison Life Sciences Building, 120 Green Street, Athens, GA 30602-7229, USA.