Quantification of adenosine-to-inosine editing of microRNAs using a conventional method

In this protocol, I describe a method for measuring the frequency of adenosine-to-inosine RNA editing of primary, precursor and mature forms of specific microRNAs (miRNAs) derived from the same source. The procedure involves reverse transcription (RT)-PCR amplification of regions containing the editing sites followed by subcloning of the PCR products and sequencing. In contrast to deep sequencing, this method does not require any specialized equipment. Pri-miRNAs, which are relatively long primary transcripts, are amplified using a conventional RT-PCR method. Therefore, this method can be adapted for any known RNA-editing sites. In contrast, 3′ polyadenylation followed by 5′ adaptor ligation is indispensable for amplification of pre-miRNAs and mature miRNAs. The complete protocol takes ∼1 week. I also include details of direct sequence analysis of the PCR products derived from pri-miRNAs as an alternative method. Although it is not as precise as the subcloning method, this procedure enables us to study RNA-editing events of many samples.

[1]  Henry Mirsky,et al.  RNA editing of a miRNA precursor. , 2004, RNA.

[2]  Hong Jiang,et al.  Identification of human fetal liver miRNAs by a novel method , 2005, FEBS letters.

[3]  Anton J. Enright,et al.  RNA editing of human microRNAs , 2006, Genome Biology.

[4]  P. Seeburg,et al.  Modulation of microRNA processing and expression through RNA editing by ADAR deaminases , 2006, Nature Structural &Molecular Biology.

[5]  C. Nusbaum,et al.  Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. , 2010, Genes & development.

[6]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[7]  K. Iwamoto,et al.  Estimating RNA editing efficiency of five editing sites in the serotonin 2C receptor by pyrosequencing. , 2005, RNA.

[8]  C. Sander,et al.  A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing , 2007, Cell.

[9]  M. Siomi,et al.  Posttranscriptional regulation of microRNA biogenesis in animals. , 2010, Molecular cell.

[10]  Bjorn-Erik Wulff,et al.  Modulation of microRNA expression and function by ADARs. , 2012, Current topics in microbiology and immunology.

[11]  R. Emeson,et al.  Quantitative analysis of 5HT2C receptor RNA editing patterns in psychiatric disorders , 2012, Neurobiology of Disease.

[12]  V. Kim,et al.  The Drosha-DGCR8 complex in primary microRNA processing. , 2004, Genes & development.

[13]  B. Bass,et al.  Adar Editing in Double-stranded Utrs and Other Noncoding Rna Sequences , 2022 .

[14]  A. Hatzigeorgiou,et al.  Redirection of Silencing Targets by Adenosine-to-Inosine Editing of miRNAs , 2007, Science.

[15]  Phillip D Zamore,et al.  microPrimer: the biogenesis and function of microRNA , 2005, Development.

[16]  G. Hannon,et al.  Processing of primary microRNAs by the Microprocessor complex , 2004, Nature.

[17]  Yukio Kawahara,et al.  RNA editing of the microRNA‐151 precursor blocks cleavage by the Dicer–TRBP complex , 2007, EMBO reports.

[18]  G. Silberberg,et al.  The edited transcriptome: novel high throughput approaches to detect nucleotide deamination. , 2011, Current opinion in genetics & development.

[19]  W. Filipowicz,et al.  Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells. , 2009, Current opinion in cell biology.

[20]  R. Shiekhattar,et al.  Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing , 2005, Cell.

[21]  R. Reenan,et al.  RNA editing in regulating gene expression in the brain. , 2008, Biochimica et biophysica acta.

[22]  S. Hammond,et al.  Emerging paradigms of regulated microRNA processing. , 2010, Genes & development.

[23]  Molly Megraw,et al.  Frequency and fate of microRNA editing in human brain , 2008, Nucleic acids research.

[24]  A. Hatzigeorgiou,et al.  Editing of Epstein-Barr Virus-encoded BART6 MicroRNAs Controls Their Dicer Targeting and Consequently Affects Viral Latency* , 2010, The Journal of Biological Chemistry.

[25]  R. Shiekhattar,et al.  The Microprocessor complex mediates the genesis of microRNAs , 2004, Nature.

[26]  D. Airey,et al.  High-Throughput Multiplexed Transcript Analysis Yields Enhanced Resolution of 5-Hydroxytryptamine2C Receptor mRNA Editing Profiles , 2010, Molecular Pharmacology.

[27]  Zefeng Wang,et al.  Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method , 2010, Nucleic acids research.

[28]  Brenda L. Bass,et al.  Predicting sites of ADAR editing in double-stranded RNA , 2011, Nature communications.

[29]  Taishin Kin,et al.  Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells , 2008, Nature.

[30]  S. Maas,et al.  Molecular diversity through RNA editing: a balancing act. , 2010, Trends in genetics : TIG.

[31]  Ammar S Naqvi,et al.  Deep annotation of Drosophila melanogaster microRNAs yields insights into their processing, modification, and emergence. , 2011, Genome research.

[32]  Victoria Arango,et al.  Altered Editing of Serotonin 2C Receptor Pre-mRNA in the Prefrontal Cortex of Depressed Suicide Victims , 2002, Neuron.