Dynamics of the Spb4 Interactome Monitored by Affinity Purification
RNA helicases constitute the largest class of NTPases involved in ribosome biogenesis, a fundamental process that has been best characterized in the eukaryotic model organism Saccharomyces cerevisiae. In yeast, genetic and biochemical analyses indicate that these RNA helicases are energy-consuming modulators of local structures inside pre-ribosomal particles that actively promote the establishment or dissociation of snoRNA:pre-rRNA base pairings, the activity of certain pre-rRNA nucleases, and/or the acquisition of pre-rRNA folds required for the recruitment or release of ribosome assembly factors and the stable assembly of ribosomal proteins. Despite significant recent advances, the precise molecular functions of RNA helicases involved in ribosome biogenesis remain largely elusive. In recent years, the purification and compositional analysis of distinct pre-ribosomal particles via affinity purification methods has been established as one of the most useful techniques to explore the yeast ribosome biogenesis pathway. In this chapter, we describe the use of different affinity purification methods to study the physical environment of RNA helicases involved in ribosome biogenesis, using as an example the putative RNA helicase Spb4 required for 60S ribosomal subunit biogenesis.
Key wordsDominant-negative mutation IgG-Sepharose precipitation GFP-trap Ribosome biogenesis RNA helicase TAP-Tandem affinity purification Yeast
We are indebted to Carine Froment and Bernard Monsarrat (Institut de Pharmacologie et de Biologie Structurale, CNRS/Université de Toulouse Paul Sabatier, France) for their valuable work on the compositional analysis of TAP-purified complexes. This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN) and ERDF (BFU2007-60151, BFU2010-15690 and HF2006-0153), and the Andalusian Government (BIO-271 and P08-CVI-03508) to J.d.l.C. and from the CNRS, Université Paul Sabatier, the Ligue Nationale Contre le Cancer (Equipe labellisée), the Agence Nationale de la Recherche, and the EGIDE Picasso Programme to Y.H.
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