Poster abstracts
Poster number 36 submitted by Allison McClish
Using GRO-Seq as a tool to understand transcriptional regulation in maize
Allison McClish (Department of Molecular Genetics), Jay Hollick (Department of Molecular Genetics; Centers for RNA Biology and Applied Plant Sciences)
Abstract:
Global Run-On Sequencing (GRO-Seq) reads identify nascent transcription occurring across the entire genome1. In maize, GRO-Seq has been used to define regions where transcription rates are dependent on RNA polymerase IV (Pol IV) function2. In Arabidopsis, Pol IV RNAs are processed into 24 nucleotide (24nt) sizes that facilitate RNA-directed DNA methylation (RdDM)3. It remains unclear in either maize or Arabidopsis whether Pol IV itself or Pol IV-dependent cytosine methylation affects Pol II function4. Comparisons of GRO-Seq profiles from dicer-like3 mutants having virtually no 24nt RNAs and rpd1 mutants having neither Pol IVa nor Pol IVb function should distinguish these two potential regulatory mechanisms. Current efforts to characterize and optimize the nuclei isolations and in vitro transcription reactions needed to create GRO-Seq libraries will be presented. Using hybridization of radiolabeled nascent RNAs with single-gene riboprobes, the effects of Sarkosyl (reported transcription initiation inhibitor5) are being evaluated and optimal elongation times are being determined. Nuclei isolated from various tissue types and developmental stages are also being evaluated for in vitro transcriptional competency and thus suitability for GRO-Seq library production. GRO-Seq profiles generated from nuclei treated with alpha-amanitin (Pol II-specific inhibitor) will potentially identify nascent Pol IV and/or Pol V transcripts. GRO-Seq profiles of specific mutants will also be compared to distinguish Pol II transcription specifically affected by Pol IVa or Pol IVb6, 7. Ultra-deep coverage of GRO-Seq reads will assist future genome annotations, including gene model validations, enhancer calls, defining 3’ pretermination transcription and other intergenic regions of non-coding transcription.
References:
1 Core et al. 2008 Science
2 Erhard et al. 2015 Genetics
3 Matzke and Mosher. 2014 Nature Reviews Genetics
4 Hale et al. 2009 PLoS Genetics
5 Gariglio et al. 1974 FEBS Letters
6 Stonaker et al. 2009 PLoS Genetics
7 Sidorenko et al. 2009 PLoS Genetics
Keywords: transcriptional regulation, RNA polymerase IV, global run-on sequencing