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: Sequencing reads were mapped to the respective reference genomes (see Supplementary Table S2) using bowtie-2 with default ‘local-sensitive’ mode (43) and further processed using samtools (44). For the small RNA libraries, reads shorter than 18 bp were discarded before mapping. To express the transcript levels for individual genes as shown in Table 1, we determined the number of reads per kilobase per million reads (RPKM) (45). Briefly, we counted the number of reads mapped to all annotated transcriptomic features (e.g. mRNA) on the same strand (i.e. sense) and opposite strand (i.e. antisense). Both the sense and antisense read numbers were normalized by length of the feature (in kilobase) and the total number of reads (in millions) mapped to non-structural RNAs in the corresponding library (i.e. the number of mappable reads excluding rRNA and tRNA reads). For the metaplot of small RNA coverage at transcription termination site (TTS), cSSRs with a TTS located at closer than 50 kb to the edge of the scaffold were discarded. To eliminate positions with abruptly high coverage, positions with coverage greater than 5-fold of the mean sense coverage upstream of the TTS were ignored. The pooled meta-coverage was then smoothed using a sliding window of 500 bp at a step size of 100 bp. The mean sense coverage upstream of a TTS was normalized to 1 within each sample. For the quantification of readthrough transcription at individual cSSRs as shown in Figure 2, the number of antisense small RNA reads per million reads (rpm) was determined within a 5-kb window downstream of the TTS. The antisense small RNA rpm was divided by the window size (5 kb) and expressed as RPKM.
Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Davison Life Sciences Building, 120 Green Street, Athens, GA 30602-7229, USA.