@article {312, title = {Small Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants.}, journal = {Plant Cell}, volume = {28}, year = {2016}, month = {2016 Feb}, pages = {286-303}, abstract = {

RNA Polymerase II (Pol II) regulatory cascades involving transcription factors (TFs) and their targets orchestrate the genetic circuitry of every eukaryotic organism. In order to understand how these cascades function, they can be dissected into small genetic networks, each containing just a few Pol II transcribed genes, that generate specific signal-processing outcomes. Small RNA regulatory circuits involve direct regulation of a small RNA by a TF and/or direct regulation of a TF by a small RNA and have been shown to play unique roles in many organisms. Here, we will focus on small RNA regulatory circuits containing Pol II transcribed microRNAs (miRNAs). While the role of miRNA-containing regulatory circuits as modular building blocks for the function of complex networks has long been on the forefront of studies in the animal kingdom, plant studies are poised to take a lead role in this area because of their advantages in probing transcriptional and posttranscriptional control of Pol II genes. The relative simplicity of tissue- and cell-type organization, miRNA targeting, and genomic structure make the Arabidopsis thaliana plant model uniquely amenable for small RNA regulatory circuit studies in a multicellular organism. In this Review, we cover analysis, tools, and validation methods for probing the component interactions in miRNA-containing regulatory circuits. We then review the important roles that plant miRNAs are playing in these circuits and summarize methods for the identification of small genetic circuits that strongly influence plant function. We conclude by noting areas of opportunity where new plant studies are imminently needed.

}, issn = {1532-298X}, doi = {10.1105/tpc.15.00852}, author = {Megraw, Molly and Cumbie, Jason S and Ivanchenko, Maria G and Filichkin, Sergei A} } @article {315, title = {Environmental stresses modulate abundance and timing of alternatively spliced circadian transcripts in Arabidopsis.}, journal = {Mol Plant}, volume = {8}, year = {2015}, month = {2015 Feb}, pages = {207-27}, abstract = {

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron-retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad-range thermal cycles triggered a sharp increase in the long IR CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or Pseudomonas syringae infection induced a similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts, whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out-of-phase oscillations of CCA1 IR transcripts. Taken together, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in\ vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of nonsense-mediated mRNA decay (NMD)-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

}, keywords = {Alternative Splicing, Arabidopsis, Arabidopsis Proteins, Circadian Clocks, Gene Expression Regulation, Plant, Introns, Nonsense Mediated mRNA Decay}, issn = {1752-9867}, doi = {10.1016/j.molp.2014.10.011}, author = {Filichkin, Sergei A and Cumbie, Jason S and Dharmawardhana, Palitha and Jaiswal, Pankaj and Chang, Jeff H and Palusa, Saiprasad G and Reddy, A S N and Megraw, Molly and Mockler, Todd C} } @article {310, title = {Improved DNase-seq protocol facilitates high resolution mapping of DNase I hypersensitive sites in roots in Arabidopsis thaliana.}, journal = {Plant Methods}, volume = {11}, year = {2015}, month = {2015}, pages = {42}, abstract = {

BACKGROUND: Identifying cis-regulatory elements is critical in understanding the direct and indirect regulatory mechanisms of gene expression. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high throughput sequencing to identify regions of regulatory DNA on a genome-wide scale. While this method was originally developed for human cell lines, later adaptations made the processing of plant tissues possible. Challenges still remain in processing recalcitrant tissues that have low DNA content.

RESULTS: By removing steps requiring the use of gel agarose plugs in DNase-seq, we were able to significantly reduce the time required to perform the protocol by at least 2\ days, while also making possible the processing of difficult plant tissues. We refer to this simplified protocol as DNase I SIM (for simplified in-nucleus method). We were able to successfully create DNase-seq libraries for both leaf and root tissues in Arabidopsis using DNase I SIM.

CONCLUSION: This protocol simplifies and facilitates generation of DNase-seq libraries from plant tissues for high resolution mapping of DNase I hypersensitive sites.

[Link to Protocol, Additional Data, and Supplementary Materials]

}, issn = {1746-4811}, doi = {10.1186/s13007-015-0087-1}, author = {Cumbie, Jason S and Filichkin, Sergei A and Megraw, Molly} } @article {317, title = {Environmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis.}, journal = {Mol Plant}, year = {2014}, month = {2014 Nov 3}, abstract = {

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad range thermal cycles triggered a sharp increase in the long intron retaining CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or P. syringae infection induced similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out of phase oscillations of CCA1 IR transcripts. Altogether, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of NMD-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.

}, issn = {1752-9867}, doi = {10.1093/mp/ssu130}, author = {Filichkin, Sergei A and Cumbie, Jason S and Dharmawadhana, J Palitha and Jaiswal, Pankaj and Chang, Jeff H and Palusa, Saiprasad G and Reddy, A S N and Megraw, Molly and Mockler, Todd C} }