Reconstitution and Biochemical Assays of an Active Human Histone Pre-mRNA 3'-end Processing Machinery

Overview

Journal Methods Enzymol

Specialty Biochemistry

Date 2021 Jun 29

PMID 34183127

Citations 3

Authors

Yadong Sun

Wei Shen Aik

Xiao-Cui Yang

William F Marzluff

Zbigniew Dominski

Liang Tong

Affiliations

Soon will be listed here.

Abstract

In animal cells, replication-dependent histone pre-mRNAs are processed at the 3'-end by an endonucleolytic cleavage carried out by the U7 snRNP, a machinery that contains the U7 snRNA and many protein subunits. Studies on the composition of this machinery and understanding of its role in 3'-end processing were greatly facilitated by the development of an in vitro system utilizing nuclear extracts from mammalian cells 35 years ago and later from Drosophila cells. Most recently, recombinant expression and purification of the components of the machinery have enabled the full reconstitution of an active machinery and its complex with a model pre-mRNA substrate, using 13 proteins and 2 RNAs, and the determination of the structure of this active machinery. This chapter presents protocols for preparing nuclear extracts containing endogenous processing machinery, for assembling semi-recombinant and fully reconstituted machineries, and for histone pre-mRNA 3'-end processing assays with these samples.

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