In Vivo Approaches to Dissecting the Function of RNA Helicases in Eukaryotic Ribosome Assembly

摘要

In eukaryotes, ribosome biogenesis involves the nucleolar transcription and processing of pre-ribosomal RNA molecules (pre-rRNA) in a complex pathway requiring the participation of myriad protein and ribonucleoprotein factors. Through efforts aimed at categorizing and characterizing these factors, at least 20 RNA helicases have been shown to interact with or participate in the activities of the major ribosome biogenesis complexes. Unfortunately, little is known about the enzymatic properties of most of these helicases, and less is known about their roles in ribosome biogenesis and pre-rRNA maturation. This chapter presents approaches for characterizing RNA helicases involved in ribosome biogenesis. Included are methods for depletion of specific protein targets, with standard protocols for assaying the typical ribosome biogenesis defects that may result. Procedures and rationales for mutagenic studies of target proteins are discussed, as well as several approaches for identifying protein–protein interactions in order to determine functional context and potential cofactors of RNA helicases.