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Nascent RNA sequencing reveals distinct features in plant transcription
Saturday, 2016/10/29 | 05:25:55
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Jonathan Hetzel, Sascha H. Duttke, Christopher Benner, and Joanne Chory SignificanceTranscription is a fundamental and dynamic step in the regulation of gene expression, but the characteristics of plant transcription are poorly understood. We adapted the global nuclear run-on sequencing (GRO-seq) and 5′GRO-seq methods for plants and provide a plant version of the next-generation sequencing software HOMER (homer.ucsd.edu/homer/plants) to facilitate data analysis. Mapping nascent transcripts in Arabidopsis thaliana seedlings enabled identification of known and novel transcripts and precisely mapped their start sites, revealing distinct characteristics in plant transcription. Our modified method to map engaged RNA polymerases and nascent transcripts in primary tissues paves the way for comparative and response studies. AbstractTranscriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5′GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved “TGT” core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole.
See http://www.pnas.org/content/113/43/12316.abstract.html?etoc PNAS October 25 2016; vol.113; no.43: 12316–12321
Fig. S2. (A) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). T4 RNAP synthesized RNA (264 nt) was kinased using T4 PNK and [α-32P]ATP or capped with the Vaccinia Capping System (M2080) and [α-32P]GTP, as described by the manufacturer. (B) Comparison of RppH activity on 32P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. (C) 32P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. (D) [32P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. (E) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on. |
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