Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Aug;10(8):1174-7.
doi: 10.1261/rna.7350304.

RNA editing of a miRNA precursor

Daniel J Luciano, Henry Mirsky, Nicholas J Vendetti, Stefan Maas

RNA editing of a miRNA precursor

Daniel J Luciano et al. RNA. 2004 Aug.

Abstract

Micro RNAs comprise a large family of small, functional RNAs with important roles in the regulation of protein coding genes in animals and plants. Here we show that human and mouse miRNA22 precursor molecules are subject to posttranscriptional modification by A-to-I RNA editing in vivo. The observed editing events are predicted to have significant implications for the biogenesis and function of miRNA22 and might point toward a more general role for RNA editing in the regulation of miRNA gene expression.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
A-to-I RNA editing of miRNA22. (A) The exon/intron structure of the human miRNA22 gene as deduced from expressed sequences with GenBank accession numbers. Exons are shown as boxes, introns as lines. The location of the mature miRNA22 sequence and the positions of the oligonucleotide primers used for editing analysis of two different splice variants A and B are indicated. (B) Predicted RNA secondary structure of pre-miRNA22 with the mature miRNA sequence underlined. Edited positions in human and/or mouse tissues (filled arrowheads) and additional sites found only in ADAR1 overexpressing cell lines (open arrowheads) are indicated. (C) Editing levels of miRNA22 precursor molecules in various human tissues, human cell lines, wild-type mouse brain, and brains of ADAR2 knock-out mice. A PCR product derived from the human miRNA22 genomic region (gDNA), as well as the reverse transcribed and amplified coding sequences from human CUTL1 (CCAAT displacement proteinlike) and DISK1 (disrupted in Schizophrenia 1) genes, were analyzed as negative controls. The number of individually sequenced clones is indicated above each column. In addition to the total extent of editing (percent of molecules edited at one or more of the 17 adenosines within the pre-miRNA), the results for the +1 position of miRNA22 are shown because they point toward a pronounced site selectivity of ADAR2 versus ADAR1. For RNA editing analysis, total RNA was isolated from human brain (Harvard Brain Tissue Collection) using Trizol Reagent (Invitrogen) and reverse transcribed with Superscript Reverse Transcriptase II (Invitrogen) and N6 random oligonucleotides as described (Maas et al. 2001). Genomic DNA was isolated from the same specimen. Additional sources for cDNA preparation were mouse brain total RNA prepared from SV129 mice, human lung and testis RNAs (Clontech), and total RNA extracted from stably transfected human cell lines (Maas et al. 2001). cDNA from ADAR2 knock-out mice (Higuchi et al. 2000) were a kind gift from Miyoko Higuchi and Peter H. Seeburg (Heidelberg). Primers miRNA22D (5′-TGAGGAGCCTGTTCCTCTCACG-3′) and miRNA22U (5′-CGCACTATGGTGCCACATCTCG-3′) for cDNA and primers gmiRNA22D (5′-GGAATTCCTTAGGAGCCTGTTCCTCTCACG-3′) and gmiRNA22U (5′-CCAGGAATCTAGAGTCTGGGC-3′) for genomic DNA were used for miRNA22-specific PCRs. Amplicons were subcloned into pBSII vector plasmid (Stratagene) and recombinant clones were isolated by blue–white selection. At least 100 individual clones were sequenced (C-tracked) from each subcloned PCR fragment. Splice form A was analyzed for all samples and splice variant B where detectable. The theoretical background of A/G discrepancies resulting from errors during reverse transcription and PCR was calculated as P = a · c + b · c · d = 3.4 · 10−5 · 17 + 0.8 · 10−5 · 17 · 24 = 0.4 · 10−2, with a being the error rate of Superscript reverse transcriptase (3.4 · 10−5; Potter et al. 2003), b the error rate of Taq DNA polymerase (0.8 · 10−5; Cline et al. 1996), c the number of adenosines analyzed, and d the number of template doublings during PCR (estimated for 30 cycles of amplification). We would therefore expect 0.4 base changes affecting adenosine in 100 sequences, corresponding to 1 base change due to mutation in 250 sequences. Because changes of A to C and A to T are also possible, this error rate is likely an overestimation because the analysis only detects A-to-G changes. The results from analysis of genomic DNA isolated from human brain is in line with this value, as within 200 sequences clones we did not detect a single A/G change. Analysis of genomic DNA from HEK293 cells yielded the same result (data not shown), as well as RT-PCR and sequencing of coding regions from human CUTL1 (BC025422) and DISC1 (AK025293) RNAs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • The use of high-throughput sequencing methods for plant microRNA research.
    Ma X, Tang Z, Qin J, Meng Y. Ma X, et al. RNA Biol. 2015;12(7):709-19. doi: 10.1080/15476286.2015.1053686. RNA Biol. 2015. PMID: 26016494 Free PMC article. Review.
  • RNA editing in the human ENCODE RNA-seq data.
    Park E, Williams B, Wold BJ, Mortazavi A. Park E, et al. Genome Res. 2012 Sep;22(9):1626-33. doi: 10.1101/gr.134957.111. Genome Res. 2012. PMID: 22955975 Free PMC article.
  • Regulation of small RNA stability: methylation and beyond.
    Ji L, Chen X. Ji L, et al. Cell Res. 2012 Apr;22(4):624-36. doi: 10.1038/cr.2012.36. Epub 2012 Mar 13. Cell Res. 2012. PMID: 22410795 Free PMC article. Review.
  • MicroRNAs and DNA damage response: implications for cancer therapy.
    Wang Y, Taniguchi T. Wang Y, et al. Cell Cycle. 2013 Jan 1;12(1):32-42. doi: 10.4161/cc.23051. Epub 2012 Dec 19. Cell Cycle. 2013. PMID: 23255103 Free PMC article. Review.
  • A mammalian microRNA expression atlas based on small RNA library sequencing.
    Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann G, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl T. Landgraf P, et al. Cell. 2007 Jun 29;129(7):1401-14. doi: 10.1016/j.cell.2007.04.040. Cell. 2007. PMID: 17604727 Free PMC article.
See all "Cited by" articles

References

    1. Bartel, D.P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116: 281–297. - PubMed
    1. Bass, B.L. 2002. RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71: 817–846. - PMC - PubMed
    1. Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–366. - PubMed
    1. Boguski, M.S., Lowe, T.M., and Tolstoshev, C.M. 1993. dbEST—Database for “expressed sequence tags.” Nat. Genet. 4: 332–333. - PubMed
    1. Burns, C.M., Chu, H., Rueter, S.M., Hutchinson, L.K., Canton, H., Sanders-Bush, E., and Emeson, R.B. 1997. Regulation of serotonin-2C receptor G-protein coupling by RNA editing [see comments]. Nature 387: 303–308. - PubMed

Publication types

LinkOut - more resources