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Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei.

Reynolds D, Cliffe L, Förstner KU, Hon CC, Siegel TN, Sabatini R - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Davison Life Sciences Building, 120 Green Street, Athens, GA 30602-7229, USA.

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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).
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Figure 1: 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).

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).


Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei.

Reynolds D, Cliffe L, Förstner KU, Hon CC, Siegel TN, Sabatini R - Nucleic Acids Res. (2014)

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).
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: 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).
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Davison Life Sciences Building, 120 Green Street, Athens, GA 30602-7229, USA.

Show MeSH
Related in: MedlinePlus