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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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RNAP II fails to terminate following reduction of base J. (A) Schematic representation of primer location and direction at a cSSR. The dashed arrow indicates readthrough transcription past the TTS. (B) Single-strand RT-PCR analysis. cDNA was synthesized using the reverse primers 2, 4, 6 and 7. PCR was performed using the same reverse primer used to make the cDNA plus a forward primer, as indicated. Three cSSRs were analyzed, two with readthrough upon J loss (22.3 and 33.5) and one without readthrough (36.3), as determined by small RNA-seq analysis. Plus RT and minus RT controls are shown. −: DMSO; +: DMOG (5 mM). (C) The amount of readthrough transcription correlates with the extent of J loss. Cells treated with 0, 1, 2.5 and 5-mM DMOG were analyzed by single-strand RT PCR as described above.
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Figure 3: RNAP II fails to terminate following reduction of base J. (A) Schematic representation of primer location and direction at a cSSR. The dashed arrow indicates readthrough transcription past the TTS. (B) Single-strand RT-PCR analysis. cDNA was synthesized using the reverse primers 2, 4, 6 and 7. PCR was performed using the same reverse primer used to make the cDNA plus a forward primer, as indicated. Three cSSRs were analyzed, two with readthrough upon J loss (22.3 and 33.5) and one without readthrough (36.3), as determined by small RNA-seq analysis. Plus RT and minus RT controls are shown. −: DMSO; +: DMOG (5 mM). (C) The amount of readthrough transcription correlates with the extent of J loss. Cells treated with 0, 1, 2.5 and 5-mM DMOG were analyzed by single-strand RT PCR as described above.

Mentions: Bloodstream form T. brucei cell line 221a of strain 427 was cultured in HMI-9 medium as described previously (40). L. major parasites were grown at 26°C in M199 media supplemented with 10% fetal bovine serum (FBS) as described (41). DMOG treatment of cells was performed by supplementing media with 1-mM DMOG for 5 days in T. brucei or at 5-mM for 10 days in L. major (1–5 mM for the DMOG titration experiments shown in Figure 3C). BrdU was supplemented into media at 10-μM or 100-μM for 6 days in L. major.


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)

RNAP II fails to terminate following reduction of base J. (A) Schematic representation of primer location and direction at a cSSR. The dashed arrow indicates readthrough transcription past the TTS. (B) Single-strand RT-PCR analysis. cDNA was synthesized using the reverse primers 2, 4, 6 and 7. PCR was performed using the same reverse primer used to make the cDNA plus a forward primer, as indicated. Three cSSRs were analyzed, two with readthrough upon J loss (22.3 and 33.5) and one without readthrough (36.3), as determined by small RNA-seq analysis. Plus RT and minus RT controls are shown. −: DMSO; +: DMOG (5 mM). (C) The amount of readthrough transcription correlates with the extent of J loss. Cells treated with 0, 1, 2.5 and 5-mM DMOG were analyzed by single-strand RT PCR as described above.
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Related In: Results  -  Collection

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Figure 3: RNAP II fails to terminate following reduction of base J. (A) Schematic representation of primer location and direction at a cSSR. The dashed arrow indicates readthrough transcription past the TTS. (B) Single-strand RT-PCR analysis. cDNA was synthesized using the reverse primers 2, 4, 6 and 7. PCR was performed using the same reverse primer used to make the cDNA plus a forward primer, as indicated. Three cSSRs were analyzed, two with readthrough upon J loss (22.3 and 33.5) and one without readthrough (36.3), as determined by small RNA-seq analysis. Plus RT and minus RT controls are shown. −: DMSO; +: DMOG (5 mM). (C) The amount of readthrough transcription correlates with the extent of J loss. Cells treated with 0, 1, 2.5 and 5-mM DMOG were analyzed by single-strand RT PCR as described above.
Mentions: Bloodstream form T. brucei cell line 221a of strain 427 was cultured in HMI-9 medium as described previously (40). L. major parasites were grown at 26°C in M199 media supplemented with 10% fetal bovine serum (FBS) as described (41). DMOG treatment of cells was performed by supplementing media with 1-mM DMOG for 5 days in T. brucei or at 5-mM for 10 days in L. major (1–5 mM for the DMOG titration experiments shown in Figure 3C). BrdU was supplemented into media at 10-μM or 100-μM for 6 days in L. major.

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