Abstract
miRNAs are a large subgroup of noncoding regulatory RNAs, which vary in length within the 20–25 nt range and show substantial length diversity and heterogeneity. To analyze the latter phenomenon, we recently developed high-resolution northern blotting and employed this method to investigate cleavages generated by recombinant human Dicer in the synthetic miRNA precursors. We paid special care to visualize clearly the cleavages generated by the individual RNase III domains of Dicer. We have compared the results of northern blotting with the results of standard analysis with the use of end-labeled RNA and visualization of Dicer cleavage products by autoradiography. The point-by-point steps of substrate preparation, recombinant Dicer cleavage assay, and northern blotting are described in this manuscript.
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References
Denli AM et al (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432:231–235
Zeng Y, Cullen BR (2005) Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J Biol Chem 280:27595–27603
Siomi H, Siomi MC (2010) Posttranscriptional regulation of microRNA biogenesis in animals. Mol Cell 38:323–332
Lund E et al (2004) Nuclear export of microRNA precursors. Science 303:95–98
Bernstein E et al (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366
Zhang H et al (2004) Single processing center models for human Dicer and bacterial RNase III. Cell 118:57–68
MacRae IJ et al (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311:195–198
Griffiths-Jones S et al (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158
Starega-Roslan J et al (2011) The role of the precursor structure in the biogenesis of microRNA. Cell Mol Life Sci 68:2859–2871
Starega-Roslan J et al (2011) Structural basis of microRNA length variety. Nucleic Acids Res 39:257–268
Landgraf P et al (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129:1401–1414
Morin RD et al (2008) Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res 18:610–621
Seitz H et al (2008) Argonaute loading improves the 5′ precision of both MicroRNAs and their miRNA* strands in flies. Curr Biol 18:147–151
Warf MB et al (2011) Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer. RNA 17:563–577
Wu H et al (2009) Alternative processing of primary microRNA transcripts by Drosha generates 5′ end variation of mature microRNA. PLoS One 4:e7566
Ruby JG et al (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127:1193–1207
Tian G et al (2010) Sequencing bias: comparison of different protocols of microRNA library construction. BMC Biotechnol 10:64
Huse SM et al (2007) Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol 8:R143
Lund E, Dahlberg JE (2006) Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs. Cold Spring Harb Symp Quant Biol 71:59–66
Maniataki E et al (2005) Immunoprecipitation of microRNPs and directional cloning of microRNAs. Methods Mol Biol 309:283–294
Leuschner PJ, Martinez J (2007) In vitro analysis of microRNA processing using recombinant Dicer and cytoplasmic extracts of HeLa cells. Methods 43:105–109
Obernosterer G et al (2006) Post-transcriptional regulation of microRNA expression. RNA 12:1161–1167
Flores-Jasso CF et al (2009) First step in pre-miRNAs processing by human Dicer. Acta Pharmacol Sin 30:1177–1185
MacRae IJ et al (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci USA 105:512–517
Koscianska E et al (2011) High-resolution northern blot for a reliable analysis of microRNAs and their precursors. Scientific World J 11:102–117
Acknowledgement
We thank W. Filipowicz for kindly providing high-purity human Dicer preps. This work was supported by the European Regional Development Fund within the Innovative Economy Programme (POIG.01.03.01-30-098/08), the European Union under the European Social Fund (8.2.2 Human Capital Operational Programme to J.S.-R.).
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Starega-Roslan, J., Krzyzosiak, W.J. (2013). Analysis of MicroRNA Length Variety Generated by Recombinant Human Dicer. In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology, vol 936. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-083-0_2
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DOI: https://doi.org/10.1007/978-1-62703-083-0_2
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