@article {745, title = {NanoCAGE-XL: An Approach to High-Confidence Transcription Start Site Sequencing}, journal = {Methods in Molecular Biology}, volume = {1830}, year = {2018}, month = {07/2018}, pages = {225-237}, abstract = {

Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5\′ 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.

}, url = {https://link.springer.com/protocol/10.1007/978-1-4939-8657-6_13}, author = {Ivanchenko, Maria G and Megraw, Molly} } @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 {316, title = {The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation.}, journal = {Development}, volume = {142}, year = {2015}, month = {2015 Feb 15}, pages = {712-21}, abstract = {

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.

}, keywords = {Arabidopsis, Biological Transport, Cyclophilin A, Indoleacetic Acids, Lycopersicon esculentum, Plant Proteins, Plant Roots, Plant Shoots}, issn = {1477-9129}, doi = {10.1242/dev.113225}, author = {Ivanchenko, Maria G and Zhu, Jinsheng and Wang, Bangjun and Medveck{\'a}, Eva and Du, Yunlong and Azzarello, Elisa and Mancuso, Stefano and Megraw, Molly and Filichkin, Sergei and Dubrovsky, Joseph G and Friml, Ji{\v r}{\'\i} and Geisler, Markus} } @article {311, title = {NanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites.}, journal = {BMC Genomics}, volume = {16}, year = {2015}, month = {2015}, pages = {597}, abstract = {

BACKGROUND: 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.

RESULTS: 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.

CONCLUSION: nanoCAGE-XL together with CapFilter allows for genome wide identification of high confidence transcription start sites in large eukaryotic genomes.

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

[Link to CapFilter Software]

}, keywords = {Arabidopsis, Genes, Plant, Genome, Plant, Nanotechnology, Plant Roots, Promoter Regions, Genetic, Sequence Analysis, DNA, Software, Transcription Initiation Site}, issn = {1471-2164}, doi = {10.1186/s12864-015-1670-6}, author = {Cumbie, Jason S and Ivanchenko, Maria G and Megraw, Molly} } @article {323, title = {Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.}, journal = {J Biol Chem}, volume = {285}, year = {2010}, month = {2010 Oct 22}, pages = {33358-70}, abstract = {

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.

}, keywords = {Cell Line, Tumor, Epstein-Barr Virus Infections, Epstein-Barr Virus Nuclear Antigens, Gene Silencing, Herpesvirus 4, Human, Humans, Immediate-Early Proteins, MicroRNAs, Ribonuclease III, RNA Editing, RNA, Viral, Trans-Activators, Viral Proteins, Virus Latency}, issn = {1083-351X}, doi = {10.1074/jbc.M110.138362}, author = {Iizasa, Hisashi and Wulff, Bjorn-Erik and Alla, Nageswara R and Maragkakis, Manolis and Megraw, Molly and Hatzigeorgiou, Artemis and Iwakiri, Dai and Takada, Kenzo and Wiedmer, Andreas and Showe, Louise and Lieberman, Paul and Nishikura, Kazuko} } @article {327, title = {Frequency and fate of microRNA editing in human brain.}, journal = {Nucleic Acids Res}, volume = {36}, year = {2008}, month = {2008 Sep}, pages = {5270-80}, abstract = {

Primary transcripts of certain microRNA (miRNA) genes (pri-miRNAs) are subject to RNA editing that converts adenosine to inosine (A--\>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--\>I editing and, thus, miRNA editing could have a large impact on the miRNA-mediated gene silencing.

}, keywords = {Adenosine, Adenosine Deaminase, Animals, Base Sequence, Brain, Humans, Inosine, Mice, MicroRNAs, Molecular Sequence Data, RNA Editing, RNA Precursors, RNA Processing, Post-Transcriptional, RNA-Binding Proteins}, issn = {1362-4962}, doi = {10.1093/nar/gkn479}, author = {Kawahara, Yukio and Megraw, Molly and Kreider, Edward and Iizasa, Hisashi and Valente, Louis and Hatzigeorgiou, Artemis G and Nishikura, Kazuko} }