Abstract
The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that have been implicated in controlling diverse pathways in a variety of organisms (1,2). For posttranscriptional gene silencing, one strand of the miRNA is used to guide components of the RNA interference machinery, including Argonaute 2, to messenger RNAs (mRNAs) with complementary sequences (3,4). Thus, targeted mRNAs are either cleaved by the endonuclease Argonaute 2 (5,6), or protein synthesis is blocked by an as yet uncharacterized mechanism (7,8). Genes encoding miRNAs are transcribed as long primary miRNAs (pri-miRNAs) that are sequentially processed by components of the nucleus and cytoplasm to yield a mature, approx 22-nucleotide (nt)-long miRNA (9). Two members of the ribonuclease (RNase) III endonuclease protein family, Drosha and Dicer, have been implicated in this two-step processing (10–13). To further our understanding of miRNA biogenesis and function it will be essential to identify the protein complexes involved. We were interested in defining the proteins required for the initial nuclear processing of pri-miRNAs to the approx 60- to 70-nt stem-loop intermediates known as precursor miRNAs (pre-miRNAs) (9,10). This led to our identification of a protein complex we termed Microprocessor, which is necessary and sufficient for processing pri-miRNA to premiRNAs (14). The Microprocessor complex comprises Drosha and the double-stranded RNAbinding protein DiGeorge syndrome critical region 8 gene (DGCR8), which is deleted in DiGeorge syndrome (15,16). In this chapter, we detail the methods used for the biochemical isolation and identification of the Microprocessor complex from human cells. We include a protocol for the in vitro analysis of pri-miRNA processing activity of the purified Microprocessor complex.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bartel D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.
Grishok A., Pasquinelli A. E., Conte D., et al. (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34.
Sontheimer E. J. (2005) Assembly and function of RNA silencing complexes. Nat. Rev. Mol. Cell Biol. 6, 127–138.
Hammond S. M., Boettcher S., Caudy A. A., Kobayashi R., and Hannon G. J. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150.
Liu J., Carmell M. A., Rivas F. V., et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.
Meister G., Landthaler M., Patkaniowska A., Dorsett Y., Teng G., and Tuschl T. (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197.
Olsen P. H. and Ambros V. (1999) The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev. Biol. 216, 671–680.
Zeng Y., Yi R., and Cullen B. R. (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc. Natl. Acad. Sci. USA 100, 9779–9784.
Lee Y., Jeon K., Lee J. T., Kim S., and Kim V. N. (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21, 4663–4670.
Lee Y., Ahn C., Han J., et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419.
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.
Ketting R. F., Fischer S. E., Bernstein E., Sijen T., Hannon G. J., and Plasterk R.H. (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659.
Hutvagner G., McLachlan J., Pasquinelli A. E., Balint E., Tuschl T., and Zamore P. D. (2001) A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838.
Gregory R. I., Yan K. P., Amuthan G., et al. (2004) The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240.
Shiohama A., Sasaki T., Noda S., Minoshima S., and Shimizu N. (2003) Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region. Biochem. Biophys. Res. Commun. 304, 184–190.
Lindsay E. A. (2001) Chromosomal microdeletions: dissecting del22q11 syndrome. Nat. Rev. Genet. 2, 58–68.
Bochar D. A., Wang L., Beniya H., et al. (2000) BRCA1 is associated with a human SWI/ SNF-related complex: linking chromatin remodeling to breast cancer. Cell 102, 257–265.
Barak O., Lazzaro M. A., Lane W. S., Speicher D. W., Picketts D. J., and Shiekhattar R. (2003) Isolation of human NURF: a regulator of Engrailed gene expression. EMBO J. 22, 6089–6100.
Dignam J. D., Lebovitz R. M., and Roeder R. G. (1983) Accurate transcription initiationby RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.s
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Gregory, R.I., Chendrimada, T.P., Shiekhattar, R. (2006). MicroRNA Biogenesis: Isolation and Characterization of the Microprocessor Complex . In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology™, vol 342. Humana Press. https://doi.org/10.1385/1-59745-123-1:33
Download citation
DOI: https://doi.org/10.1385/1-59745-123-1:33
Publisher Name: Humana Press
Print ISBN: 978-1-58829-581-1
Online ISBN: 978-1-59745-123-9
eBook Packages: Springer Protocols