Purification of Microprocessor-Associated Factors

Ross A Cordiner; Sara Macias

doi:10.1007/978-1-4939-8624-8_5

Purification of Microprocessor-Associated Factors

Methods Mol Biol. 2018:1823:51-62. doi: 10.1007/978-1-4939-8624-8_5.

Authors

Ross A Cordiner¹, Sara Macias²

Affiliations

¹ Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology and Genetics, Aarhus University, C. F. Møllers Alle 3, Bldg. 1130, 8000 Aarhus C, Aarhus, Denmark.
² Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3FL, UK. sara.maciasribela@ed.ac.uk.

PMID: 29959673
DOI: 10.1007/978-1-4939-8624-8_5

Abstract

The Microprocessor complex catalyzes the first step of miRNA biogenesis in the nucleus of mammalian cells. The minimal catalytically active complex is formed by two essential factors, the dsRNA binding protein DGCR8, and the RNase III endonuclease Drosha. Importantly, several co-factors can associate to this complex and modulate the cleavage and binding efficiency of this complex, in a positive or negative manner. Here, we describe a simple method for purification of DGCR8 and Drosha coupled to mass spectrometry or western blot which allows robust identification of unknown associated factors. This approach has recently revealed the presence of a new DGCR8-dependent, Drosha-independent complex involved in RNA turnover.

Keywords: DGCR8; Drosha; Immunoprecipitation; Mass spectrometry; Pri-miRNA processing; RNA processing; Western blot; miRNAs.

MeSH terms

Cell Nucleus / chemistry*
Cell Nucleus / metabolism
HeLa Cells
Humans
Mass Spectrometry / methods
RNA-Binding Proteins / chemistry*
RNA-Binding Proteins / isolation & purification*
RNA-Binding Proteins / metabolism
Ribonuclease III / chemistry*
Ribonuclease III / isolation & purification*

Substances

DGCR8 protein, human
RNA-Binding Proteins
DROSHA protein, human
Ribonuclease III