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

doi:10.1016/bs.mie.2021.03.021

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

Department of Chemistry

Research output: Chapter in book/report/conference proceeding › Chapter › peer-review

3 Citations (Scopus)

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 language	English
Title of host publication	mRNA 3' End Processing and Metabolism
Editors	Bin Tian
Publisher	Academic Press Inc.
Pages	291-324
Number of pages	34
Edition	1st
ISBN (Electronic)	9780128235744
ISBN (Print)	9780128235737
DOIs	https://doi.org/10.1016/bs.mie.2021.03.021
Publication status	Published - 25 Jun 2021

Publication series

Name	Methods in Enzymology
Volume	655
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

Access to Document

10.1016/bs.mie.2021.03.021

Cite this

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

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.",

keywords = "Cleavage and polyadenylation, CPSF73, Endonuclease, Exonuclease, Fluorescence nuclease assays, Nuclear extract, Radioactive nuclease assays",

author = "Yadong Sun and Aik, {Wei Shen} and Yang, {Xiao Cui} and Marzluff, {William F.} and Zbigniew Dominski and Liang Tong",

note = "Funding Information: This research is supported by NIH grants R35GM118093 (to LT) and R01GM029832 (to WFM and ZD). WSA was also supported by a fellowship from the Raymond and Beverley Sackler Center for Research at Convergence of Disciplines at Columbia University Medical Center.",

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doi = "10.1016/bs.mie.2021.03.021",

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series = "Methods in Enzymology",

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Reconstitution and biochemical assays of an active human histone pre-mRNA 3′-end processing machinery. / Sun, Yadong; Aik, Wei Shen; Yang, Xiao Cui et al.
mRNA 3' End Processing and Metabolism. ed. / Bin Tian. 1st. ed. Academic Press Inc., 2021. p. 291-324 (Methods in Enzymology; Vol. 655).

Research output: Chapter in book/report/conference proceeding › Chapter › peer-review

TY - CHAP

T1 - Reconstitution and biochemical assays of an active human histone pre-mRNA 3′-end processing machinery

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AU - Aik, Wei Shen

AU - Yang, Xiao Cui

AU - Marzluff, William F.

AU - Dominski, Zbigniew

AU - Tong, Liang

N1 - Funding Information: This research is supported by NIH grants R35GM118093 (to LT) and R01GM029832 (to WFM and ZD). WSA was also supported by a fellowship from the Raymond and Beverley Sackler Center for Research at Convergence of Disciplines at Columbia University Medical Center.

PY - 2021/6/25

Y1 - 2021/6/25

N2 - 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.

AB - 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.

KW - Cleavage and polyadenylation

KW - CPSF73

KW - Endonuclease

KW - Exonuclease

KW - Fluorescence nuclease assays

KW - Nuclear extract

KW - Radioactive nuclease assays

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SN - 9780128235737

T3 - Methods in Enzymology

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BT - mRNA 3' End Processing and Metabolism

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PB - Academic Press Inc.

ER -

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

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