01333nas a2200145   4500008004100000245008400041210006900125260001200194300001200206490000900218520084900227100002501076700001801101856006801119       2018                        eng d00aNanoCAGE-XL: An Approach to High-Confidence Transcription Start Site Sequencing0 aNanoCAGEXL An Approach to HighConfidence Transcription Start Sit  c07/2018  a225-2370 v18303 a<p>Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5&prime; 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 this limitation. In this chapter, we present the nanoCAGE-XL protocol, the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high-throughput platforms such as Illumina HiSeq-2000. NanoCAGE-XL provides a method for precise transcription start site identification in large eukaryotic genomes, even in cases where input total RNA quantity is very limited.</p>1 aIvanchenko, Maria, G1 aMegraw, Molly  uhttps://link.springer.com/protocol/10.1007/978-1-4939-8657-6_1302264nas 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/31202854nas a2200373   4500008004100000022001400041245012300055210006900178260001600247300001100263490000800274520174600282653001602028653002502044653001802069653002302087653002802110653001902138653001602157653001702173100002502190700001802215700001802233700001902251700001602270700002102286700002102307700001802328700002202346700002502368700001802393700002002411856004902431       2015                        eng d  a1477-912900aThe cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation.0 acyclophilin A DIAGEOTROPICA gene affects auxin transport in both  c2015 Feb 15  a712-210 v1423 a<p>Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion restores the process of lateral root formation, consistent with participation of a mobile signal. Antibodies do not detect movement of the DGT protein into the dgt rootstock; however, experiments with radiolabeled auxin and an auxin-specific microelectrode demonstrate abnormal auxin fluxes. Functional studies of DGT in heterologous yeast and tobacco-leaf auxin-transport systems demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin efflux transporters by affecting their plasma membrane localization. Studies in tomato support complex effects of the dgt mutation on PIN expression level, expression domain and plasma membrane localization. Our data demonstrate that DGT regulates auxin transport in lateral root formation.</p>10aArabidopsis10aBiological Transport10aCyclophilin A10aIndoleacetic Acids10aLycopersicon esculentum10aPlant Proteins10aPlant Roots10aPlant Shoots1 aIvanchenko, Maria, G1 aZhu, Jinsheng1 aWang, Bangjun1 aMedvecká, Eva1 aDu, Yunlong1 aAzzarello, Elisa1 aMancuso, Stefano1 aMegraw, Molly1 aFilichkin, Sergei1 aDubrovsky, Joseph, G1 aFriml, Jiří1 aGeisler, Markus  uhttps://megraw.cgrb.oregonstate.edu/node/31602178nas 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/31102616nas a2200445   4500008004100000022001400041245012900055210006900184260001600253300001300269490000800282520127800290653002101568653003401589653004001623653001901663653002501682653001101707653002901718653001401747653002101761653001601782653001501798653002101813653001901834653001801853100002001871700002201891700002301913700002401936700001801960700002701978700001702005700001802022700002102040700001802061700002002079700002202099856004902121       2010                        eng d  a1083-351X00aEditing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.0 aEditing of EpsteinBarr virusencoded BART6 microRNAs controls the  c2010 Oct 22  a33358-700 v2853 a<p>Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3&#39;-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.</p>10aCell Line, Tumor10aEpstein-Barr Virus Infections10aEpstein-Barr Virus Nuclear Antigens10aGene Silencing10aHerpesvirus 4, Human10aHumans10aImmediate-Early Proteins10aMicroRNAs10aRibonuclease III10aRNA Editing10aRNA, Viral10aTrans-Activators10aViral Proteins10aVirus Latency1 aIizasa, Hisashi1 aWulff, Bjorn-Erik1 aAlla, Nageswara, R1 aMaragkakis, Manolis1 aMegraw, Molly1 aHatzigeorgiou, Artemis1 aIwakiri, Dai1 aTakada, Kenzo1 aWiedmer, Andreas1 aShowe, Louise1 aLieberman, Paul1 aNishikura, Kazuko  uhttps://megraw.cgrb.oregonstate.edu/node/32302435nas a2200385   4500008004100000022001400041245005900055210005800114260001300172300001200185490000700197520139400204653001401598653002401612653001201636653001801648653001001666653001101676653001201687653000901699653001401708653002801722653001601750653001901766653004101785653002501826100002001851700001801871700002001889700002001909700001901929700003001948700002201978856004902000       2008                        eng d  a1362-496200aFrequency and fate of microRNA editing in human brain.0 aFrequency and fate of microRNA editing in human brain  c2008 Sep  a5270-800 v363 a<p>Primary transcripts of certain microRNA (miRNA) genes (pri-miRNAs) are subject to RNA editing that converts adenosine to inosine (A--&gt;I RNA editing). However, the frequency of the pri-miRNA editing and the fate of edited pri-miRNAs remain largely to be determined. Examination of already known pri-miRNA editing sites indicated that adenosine residues of the UAG triplet sequence might be edited more frequently. In the present study, therefore, we conducted a large-scale survey of human pri-miRNAs containing the UAG triplet sequence. By direct sequencing of RT-PCR products corresponding to pri-miRNAs, we examined 209 pri-miRNAs and identified 43 UAG and also 43 non-UAG editing sites in 47 pri-miRNAs, which were highly edited in human brain. In vitro miRNA processing assay using recombinant Drosha-DGCR8 and Dicer-TRBP (the human immuno deficiency virus transactivating response RNA-binding protein) complexes revealed that a majority of pri-miRNA editing is likely to interfere with the miRNA processing steps. In addition, four new edited miRNAs with altered seed sequences were identified by targeted cloning and sequencing of the miRNAs that would be processed from edited pri-miRNAs. Our studies predict that approximately 16% of human pri-miRNAs are subject to A--&gt;I editing and, thus, miRNA editing could have a large impact on the miRNA-mediated gene silencing.</p>10aAdenosine10aAdenosine Deaminase10aAnimals10aBase Sequence10aBrain10aHumans10aInosine10aMice10aMicroRNAs10aMolecular Sequence Data10aRNA Editing10aRNA Precursors10aRNA Processing, Post-Transcriptional10aRNA-Binding Proteins1 aKawahara, Yukio1 aMegraw, Molly1 aKreider, Edward1 aIizasa, Hisashi1 aValente, Louis1 aHatzigeorgiou, Artemis, G1 aNishikura, Kazuko  uhttps://megraw.cgrb.oregonstate.edu/node/327