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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 does not lead to readthrough transcription in T. brucei at cSSRs. (A) Anti-base J dot blot analysis of WT T. brucei was performed as detailed for Figure 1A, but T. brucei genomic DNA was isolated from cells treated with either 1-mM DMOG or DMSO for 5 days. (B) Anti-base J IP qPCR analysis. Same as shown in Figure 1B, where white bars show % IP for DMSO and black bars show % J IP from DMOG-treated T. brucei DNA. Four cSSRs enriched for base J are shown. Error bars represent the standard deviation. (C) Small RNA-seq analysis of three cSSRs (10.8, 10.3 and 11.9) is shown with reads plotted as reads per million reads mapped (rpm). Top graphs: DMSO-treated WT; bottom graphs: DMOG-treated WT. Reads mapped to the top strand are shown in blue and reads mapped to the bottom strand in red. ORFs and their chromosomal location in kb are shown above. (D) A metaplot summarizing the readthrough defect at cSSRs (n = 87, 11 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). (E) Confirmation of total RNA-seq transcript changes in T. brucei by RT qPCR. Fold change in transcript abundance, with DMSO-treated WT set to 1. Striped bars: fold change based on RT qPCR analysis of DMOG-treated WT T. brucei; gray bars: fold change in the RPKM of DMOG-treated WT T. brucei based on total RNA-seq analysis. For RT qPCR analysis, transcripts were normalized against 40s rRNA. Transcripts analyzed included 3230 (Tb427.07.3230), which did not change in abundance after DMOG treatment; 1660 (Tb427.08.1660), 2590 (Tb427.03.2590), 4930 (Tb427tmp.160.4930) and 2000 (Tb427.07.2000), which were increased by at least 2-fold after DMOG treatment; and 10230 (Tb427.10.10230), which was decreased about 2-fold following DMOG treatment. Error bars represent the standard deviation of three independent biological replicates.
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Figure 4: Loss of base J does not lead to readthrough transcription in T. brucei at cSSRs. (A) Anti-base J dot blot analysis of WT T. brucei was performed as detailed for Figure 1A, but T. brucei genomic DNA was isolated from cells treated with either 1-mM DMOG or DMSO for 5 days. (B) Anti-base J IP qPCR analysis. Same as shown in Figure 1B, where white bars show % IP for DMSO and black bars show % J IP from DMOG-treated T. brucei DNA. Four cSSRs enriched for base J are shown. Error bars represent the standard deviation. (C) Small RNA-seq analysis of three cSSRs (10.8, 10.3 and 11.9) is shown with reads plotted as reads per million reads mapped (rpm). Top graphs: DMSO-treated WT; bottom graphs: DMOG-treated WT. Reads mapped to the top strand are shown in blue and reads mapped to the bottom strand in red. ORFs and their chromosomal location in kb are shown above. (D) A metaplot summarizing the readthrough defect at cSSRs (n = 87, 11 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). (E) Confirmation of total RNA-seq transcript changes in T. brucei by RT qPCR. Fold change in transcript abundance, with DMSO-treated WT set to 1. Striped bars: fold change based on RT qPCR analysis of DMOG-treated WT T. brucei; gray bars: fold change in the RPKM of DMOG-treated WT T. brucei based on total RNA-seq analysis. For RT qPCR analysis, transcripts were normalized against 40s rRNA. Transcripts analyzed included 3230 (Tb427.07.3230), which did not change in abundance after DMOG treatment; 1660 (Tb427.08.1660), 2590 (Tb427.03.2590), 4930 (Tb427tmp.160.4930) and 2000 (Tb427.07.2000), which were increased by at least 2-fold after DMOG treatment; and 10230 (Tb427.10.10230), which was decreased about 2-fold following DMOG treatment. Error bars represent the standard deviation of three independent biological replicates.

Mentions: The termination defects observed in Leishmania spp. prompted us to examine T. brucei for similar transcription termination defects upon the loss of base J. Base J is not essential in T. brucei, as both JBP enzymes can be knocked out without obvious phenotypic effects (34). Consistent with this, wild-type T. brucei treated with 1-mM DMOG reduced global J levels beyond the limits of detection by anti-J dot blot (Figure 4A), with no significant growth defect (Supplementary Figure S4). Consistent with total J levels, all cSSRs examined had significantly reduced levels of J following DMOG treatment (Figure 4B).


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 does not lead to readthrough transcription in T. brucei at cSSRs. (A) Anti-base J dot blot analysis of WT T. brucei was performed as detailed for Figure 1A, but T. brucei genomic DNA was isolated from cells treated with either 1-mM DMOG or DMSO for 5 days. (B) Anti-base J IP qPCR analysis. Same as shown in Figure 1B, where white bars show % IP for DMSO and black bars show % J IP from DMOG-treated T. brucei DNA. Four cSSRs enriched for base J are shown. Error bars represent the standard deviation. (C) Small RNA-seq analysis of three cSSRs (10.8, 10.3 and 11.9) is shown with reads plotted as reads per million reads mapped (rpm). Top graphs: DMSO-treated WT; bottom graphs: DMOG-treated WT. Reads mapped to the top strand are shown in blue and reads mapped to the bottom strand in red. ORFs and their chromosomal location in kb are shown above. (D) A metaplot summarizing the readthrough defect at cSSRs (n = 87, 11 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). (E) Confirmation of total RNA-seq transcript changes in T. brucei by RT qPCR. Fold change in transcript abundance, with DMSO-treated WT set to 1. Striped bars: fold change based on RT qPCR analysis of DMOG-treated WT T. brucei; gray bars: fold change in the RPKM of DMOG-treated WT T. brucei based on total RNA-seq analysis. For RT qPCR analysis, transcripts were normalized against 40s rRNA. Transcripts analyzed included 3230 (Tb427.07.3230), which did not change in abundance after DMOG treatment; 1660 (Tb427.08.1660), 2590 (Tb427.03.2590), 4930 (Tb427tmp.160.4930) and 2000 (Tb427.07.2000), which were increased by at least 2-fold after DMOG treatment; and 10230 (Tb427.10.10230), which was decreased about 2-fold following DMOG treatment. Error bars represent the standard deviation of three independent biological replicates.
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Figure 4: Loss of base J does not lead to readthrough transcription in T. brucei at cSSRs. (A) Anti-base J dot blot analysis of WT T. brucei was performed as detailed for Figure 1A, but T. brucei genomic DNA was isolated from cells treated with either 1-mM DMOG or DMSO for 5 days. (B) Anti-base J IP qPCR analysis. Same as shown in Figure 1B, where white bars show % IP for DMSO and black bars show % J IP from DMOG-treated T. brucei DNA. Four cSSRs enriched for base J are shown. Error bars represent the standard deviation. (C) Small RNA-seq analysis of three cSSRs (10.8, 10.3 and 11.9) is shown with reads plotted as reads per million reads mapped (rpm). Top graphs: DMSO-treated WT; bottom graphs: DMOG-treated WT. Reads mapped to the top strand are shown in blue and reads mapped to the bottom strand in red. ORFs and their chromosomal location in kb are shown above. (D) A metaplot summarizing the readthrough defect at cSSRs (n = 87, 11 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). (E) Confirmation of total RNA-seq transcript changes in T. brucei by RT qPCR. Fold change in transcript abundance, with DMSO-treated WT set to 1. Striped bars: fold change based on RT qPCR analysis of DMOG-treated WT T. brucei; gray bars: fold change in the RPKM of DMOG-treated WT T. brucei based on total RNA-seq analysis. For RT qPCR analysis, transcripts were normalized against 40s rRNA. Transcripts analyzed included 3230 (Tb427.07.3230), which did not change in abundance after DMOG treatment; 1660 (Tb427.08.1660), 2590 (Tb427.03.2590), 4930 (Tb427tmp.160.4930) and 2000 (Tb427.07.2000), which were increased by at least 2-fold after DMOG treatment; and 10230 (Tb427.10.10230), which was decreased about 2-fold following DMOG treatment. Error bars represent the standard deviation of three independent biological replicates.
Mentions: The termination defects observed in Leishmania spp. prompted us to examine T. brucei for similar transcription termination defects upon the loss of base J. Base J is not essential in T. brucei, as both JBP enzymes can be knocked out without obvious phenotypic effects (34). Consistent with this, wild-type T. brucei treated with 1-mM DMOG reduced global J levels beyond the limits of detection by anti-J dot blot (Figure 4A), with no significant growth defect (Supplementary Figure S4). Consistent with total J levels, all cSSRs examined had significantly reduced levels of J following DMOG treatment (Figure 4B).

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