02264nas a2200181   4500008004100000022001400041245010600055210006900161260001300230300001200243490000700255520168200262100001801944700002101962700002501983700002502008856004902033       2016                        eng d  a1532-298X00aSmall Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants.0 aSmall Genetic Circuits and MicroRNAs Big Players in Polymerase I  c2016 Feb  a286-3030 v283 a<p>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.</p>1 aMegraw, Molly1 aCumbie, Jason, S1 aIvanchenko, Maria, G1 aFilichkin, Sergei, A  uhttps://megraw.cgrb.oregonstate.edu/node/31202503nas a2200325   4500008004100000022001400041245012000055210006900175260001300244300001100257490000600268520148900274653002501763653001601788653002501804653002101829653003801850653001201888653003301900100002501933700002101958700002801979700002002007700001902027700002502046700001802071700001802089700002102107856004902128       2015                        eng d  a1752-986700aEnvironmental stresses modulate abundance and timing of alternatively spliced circadian transcripts in Arabidopsis.0 aEnvironmental stresses modulate abundance and timing of alternat  c2015 Feb  a207-270 v83 a<p>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&nbsp;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.</p>10aAlternative Splicing10aArabidopsis10aArabidopsis Proteins10aCircadian Clocks10aGene Expression Regulation, Plant10aIntrons10aNonsense Mediated mRNA Decay1 aFilichkin, Sergei, A1 aCumbie, Jason, S1 aDharmawardhana, Palitha1 aJaiswal, Pankaj1 aChang, Jeff, H1 aPalusa, Saiprasad, G1 aReddy, A, S N1 aMegraw, Molly1 aMockler, Todd, C  uhttps://megraw.cgrb.oregonstate.edu/node/31501941nas a2200169   4500008004100000022001400041245013400055210006900189260000900258300000700267490000700274520137700281100002101658700002501679700001801704856004901722       2015                        eng d  a1746-481100aImproved DNase-seq protocol facilitates high resolution mapping of DNase I hypersensitive sites in roots in Arabidopsis thaliana.0 aImproved DNaseseq protocol facilitates high resolution mapping o  c2015  a420 v113 a<p><b>BACKGROUND: </b>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.</p><p><b>RESULTS: </b>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&nbsp;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.</p><p><b>CONCLUSION: </b>This protocol simplifies and facilitates generation of DNase-seq libraries from plant tissues for high resolution mapping of DNase I hypersensitive sites.</p><p><a href="http://megraw-dev.cgrb.oregonstate.edu/DNaseISIM">[Link to Protocol, Additional Data, and Supplementary Materials]</a></p>1 aCumbie, Jason, S1 aFilichkin, Sergei, A1 aMegraw, Molly  uhttps://megraw.cgrb.oregonstate.edu/node/31002178nas a2200277   4500008004100000022001400041245011900055210006900174260000900243300000800252490000700260520133000267653001601597653001701613653001801630653001901648653001601667653003001683653002701713653001301740653003401753100002101787700002501808700001801833856004901851       2015                        eng d  a1471-216400aNanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites.0 aNanoCAGEXL and CapFilter an approach to genome wide identificati  c2015  a5970 v163 a<p><b>BACKGROUND: </b>Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5&#39; end of transcripts via Cap Analysis of Gene Expression (CAGE) or linker-ligation strategies such as Paired-End Analysis of Transcription Start Sites (PEAT), but often require large amounts of tissue. More recently, nanoCAGE was developed for sequencing on the Illumina GAIIx to overcome these difficulties.</p><p><b>RESULTS: </b>Here we present the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high throughput platforms such as Illumina HiSeq-2000, and CapFilter, a computational pipeline that greatly increases confidence in TSS identification. We report excellent gene coverage, reproducibility, and precision in transcription start site discovery for samples from Arabidopsis thaliana roots.</p><p><b>CONCLUSION: </b>nanoCAGE-XL together with CapFilter allows for genome wide identification of high confidence transcription start sites in large eukaryotic genomes.</p><p><a href="http://megraw-dev.cgrb.oregonstate.edu/nanocage">[Link to Protocol, Additional Data, and Supplementary Materials]</a></p><p><a href="http://megraw-dev.cgrb.oregonstate.edu/capfilter">[Link to CapFilter Software]</a></p>10aArabidopsis10aGenes, Plant10aGenome, Plant10aNanotechnology10aPlant Roots10aPromoter Regions, Genetic10aSequence Analysis, DNA10aSoftware10aTranscription Initiation Site1 aCumbie, Jason, S1 aIvanchenko, Maria, G1 aMegraw, Molly  uhttps://megraw.cgrb.oregonstate.edu/node/31102171nas a2200217   4500008004100000022001400041245012000055210006900175260001500244520145100259100002501710700002101735700002701756700002001783700001901803700002501822700001801847700001801865700002101883856004901904       2014                        eng d  a1752-986700aEnvironmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis.0 aEnvironmental Stresses Modulate Abundance and Timing of Alternat  c2014 Nov 33 a<p>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.</p>1 aFilichkin, Sergei, A1 aCumbie, Jason, S1 aDharmawadhana, Palitha1 aJaiswal, Pankaj1 aChang, Jeff, H1 aPalusa, Saiprasad, G1 aReddy, A, S N1 aMegraw, Molly1 aMockler, Todd, C  uhttps://megraw.cgrb.oregonstate.edu/node/317