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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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Nuclear run-on analysis of the region shown in Figure 5. (A) The location of probes A–F is shown. (B) Results from a representative experiment are shown, with DMSO-treated WT on the left and DMOG-treated WT on the right. The probes used include SL: spliced leader; Tub: tubulin; Eno: enolase; 5.8S: 5.8S rRNA; 5S: 5S rRNA; 18.5S: 18.5S rRNA; −: empty pCR2.1 vector; and probes A–F illustrated above. The radioactive signal was measured using a phosphorimager and normalized using the SL signal. The empty pCR2.1 vector was used to subtract background signal. (C) The normalized signal—white bars: DMSO; black bars: DMOG. (D) Fold change in the DMOG-treated WT signal, with the DMSO-treated WT signal set to 1.
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Figure 6: Nuclear run-on analysis of the region shown in Figure 5. (A) The location of probes A–F is shown. (B) Results from a representative experiment are shown, with DMSO-treated WT on the left and DMOG-treated WT on the right. The probes used include SL: spliced leader; Tub: tubulin; Eno: enolase; 5.8S: 5.8S rRNA; 5S: 5S rRNA; 18.5S: 18.5S rRNA; −: empty pCR2.1 vector; and probes A–F illustrated above. The radioactive signal was measured using a phosphorimager and normalized using the SL signal. The empty pCR2.1 vector was used to subtract background signal. (C) The normalized signal—white bars: DMSO; black bars: DMOG. (D) Fold change in the DMOG-treated WT signal, with the DMSO-treated WT signal set to 1.

Mentions: Interestingly, 18 out of the 27 upregulated genes (67%) are located downstream of a J peak within a gene cluster, suggesting that base J may facilitate termination prior to the end of a gene cluster and attenuate the transcription of downstream genes. Consistent with RNAP II termination prior to the end of a PTU, H3V is also enriched upstream of many of the upregulated genes (5). An example is shown in Figure 5, where a peak of base J and H3V is found upstream of the last two genes in a PTU. Both of the downstream genes are lowly expressed in the presence of base J, but increased upon the loss of J (Figure 5A–C) (for additional examples see Supplementary Figure S7). As described above, removal of DMOG restored base J synthesis and wild-type expression levels (Figure 5D and E). To examine whether the upregulation was due to increased transcription, as opposed to post-transcriptional mechanisms, we performed nuclear run-ons. Consistent with transcription termination occurring prior to the last two genes in this gene cluster, the signal from elongating RNAP is lost between the region covered by probes C and D in WT cells (Figure 6). Termination in this region is further supported by the RNA-seq data and localization of base J and H3V (Figure 5A–C). Upon the loss of base J, we found increased transcription of the region downstream of the peak of J (Figure 6 and Supplementary Figure S8), suggesting that increases in mRNA abundance for these two genes occurred at the level of transcription. Further support for readthrough transcription at this site is provided by single-strand RT-PCR analysis (Supplementary Figure S9). Following the loss of base J we detect an increase in a nascent transcript that extends downstream of the proposed termination site. Overall these results indicate that while base J may not regulate RNAP II termination in T. brucei at previously defined sites at cSSRs and HT regions, it may attenuate transcription elongation within specific PTUs and enable regulated expression of downstream genes.


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)

Nuclear run-on analysis of the region shown in Figure 5. (A) The location of probes A–F is shown. (B) Results from a representative experiment are shown, with DMSO-treated WT on the left and DMOG-treated WT on the right. The probes used include SL: spliced leader; Tub: tubulin; Eno: enolase; 5.8S: 5.8S rRNA; 5S: 5S rRNA; 18.5S: 18.5S rRNA; −: empty pCR2.1 vector; and probes A–F illustrated above. The radioactive signal was measured using a phosphorimager and normalized using the SL signal. The empty pCR2.1 vector was used to subtract background signal. (C) The normalized signal—white bars: DMSO; black bars: DMOG. (D) Fold change in the DMOG-treated WT signal, with the DMSO-treated WT signal set to 1.
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Figure 6: Nuclear run-on analysis of the region shown in Figure 5. (A) The location of probes A–F is shown. (B) Results from a representative experiment are shown, with DMSO-treated WT on the left and DMOG-treated WT on the right. The probes used include SL: spliced leader; Tub: tubulin; Eno: enolase; 5.8S: 5.8S rRNA; 5S: 5S rRNA; 18.5S: 18.5S rRNA; −: empty pCR2.1 vector; and probes A–F illustrated above. The radioactive signal was measured using a phosphorimager and normalized using the SL signal. The empty pCR2.1 vector was used to subtract background signal. (C) The normalized signal—white bars: DMSO; black bars: DMOG. (D) Fold change in the DMOG-treated WT signal, with the DMSO-treated WT signal set to 1.
Mentions: Interestingly, 18 out of the 27 upregulated genes (67%) are located downstream of a J peak within a gene cluster, suggesting that base J may facilitate termination prior to the end of a gene cluster and attenuate the transcription of downstream genes. Consistent with RNAP II termination prior to the end of a PTU, H3V is also enriched upstream of many of the upregulated genes (5). An example is shown in Figure 5, where a peak of base J and H3V is found upstream of the last two genes in a PTU. Both of the downstream genes are lowly expressed in the presence of base J, but increased upon the loss of J (Figure 5A–C) (for additional examples see Supplementary Figure S7). As described above, removal of DMOG restored base J synthesis and wild-type expression levels (Figure 5D and E). To examine whether the upregulation was due to increased transcription, as opposed to post-transcriptional mechanisms, we performed nuclear run-ons. Consistent with transcription termination occurring prior to the last two genes in this gene cluster, the signal from elongating RNAP is lost between the region covered by probes C and D in WT cells (Figure 6). Termination in this region is further supported by the RNA-seq data and localization of base J and H3V (Figure 5A–C). Upon the loss of base J, we found increased transcription of the region downstream of the peak of J (Figure 6 and Supplementary Figure S8), suggesting that increases in mRNA abundance for these two genes occurred at the level of transcription. Further support for readthrough transcription at this site is provided by single-strand RT-PCR analysis (Supplementary Figure S9). Following the loss of base J we detect an increase in a nascent transcript that extends downstream of the proposed termination site. Overall these results indicate that while base J may not regulate RNAP II termination in T. brucei at previously defined sites at cSSRs and HT regions, it may attenuate transcription elongation within specific PTUs and enable regulated expression of downstream genes.

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