Reconstitution and biochemical assays of an active human histone pre-mRNA 3′-end processing machinery

Yadong Sun, Wei Shen AIK, Xiao Cui Yang, William F. Marzluff, Zbigniew Dominski*, Liang Tong

*Corresponding author for this work

Research output: Chapter in book/report/conference proceedingChapterpeer-review

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.

Original languageEnglish
Title of host publicationmRNA 3' End Processing and Metabolism
EditorsBin Tian
PublisherAcademic Press Inc.
Pages291-324
Number of pages34
ISBN (Print)9780128235737
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Enzymology
Volume655
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Scopus Subject Areas

  • Biochemistry
  • Molecular Biology

User-Defined Keywords

  • Cleavage and polyadenylation
  • CPSF73
  • Endonuclease
  • Exonuclease
  • Fluorescence nuclease assays
  • Nuclear extract
  • Radioactive nuclease assays

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