Isolation of RNP granules

Lars Jønson; Finn Cilius Nielsen; Jan Christiansen

doi:10.1007/978-1-59745-248-9_18

Isolation of RNP granules

Lars Jønson, Finn Cilius Nielsen, Jan Christiansen

Department of Clinical Biochemistry

2 Citations (Scopus)

Abstract

The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, mRNAs. The RNP granule can be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient "handle" for the subsequent immunoprecipitation by immobilized anti-FLAG antibody.

Original language	English
Journal	Methods in Molecular Biology
Volume	703
Pages (from-to)	265-73
Number of pages	9
ISSN	1064-3745
DOIs	https://doi.org/10.1007/978-1-59745-248-9_18
Publication status	Published - 2011

Keywords

Blotting, Western
Cytoplasmic Granules
Electrophoresis, Polyacrylamide Gel
Gene Expression Regulation
Immunoprecipitation
RNA, Messenger
RNA-Binding Proteins
Ribonucleoproteins
Tetracycline

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10.1007/978-1-59745-248-9_18

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title = "Isolation of RNP granules",

abstract = "The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, mRNAs. The RNP granule can be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient {"}handle{"} for the subsequent immunoprecipitation by immobilized anti-FLAG antibody.",

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year = "2011",

doi = "10.1007/978-1-59745-248-9\_18",

language = "English",

volume = "703",

pages = "265--73",

journal = "Methods in Molecular Biology",

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publisher = "Humana Press Inc.",

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TY - JOUR

T1 - Isolation of RNP granules

AU - Jønson, Lars

AU - Nielsen, Finn Cilius

AU - Christiansen, Jan

PY - 2011

Y1 - 2011

N2 - The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, mRNAs. The RNP granule can be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient "handle" for the subsequent immunoprecipitation by immobilized anti-FLAG antibody.

AB - The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, mRNAs. The RNP granule can be regarded as a supramolecular assembly of RNA and protein, probably representing several overlapping post-transcriptional operons. The present protocol describes how RNP granules may be isolated by the transgenic expression of a 3X FLAG version of an RNA-binding protein under tetracycline control via the tetracycline receptor/operator complex. In this way, inclusion of an appropriate tetracycline concentration ensures expression of the tagged version at the endogenous level, and the 3X FLAG tag is a convenient "handle" for the subsequent immunoprecipitation by immobilized anti-FLAG antibody.

KW - Blotting, Western

KW - Cytoplasmic Granules

KW - Electrophoresis, Polyacrylamide Gel

KW - Gene Expression Regulation

KW - Immunoprecipitation

KW - RNA, Messenger

KW - RNA-Binding Proteins

KW - Ribonucleoproteins

KW - Tetracycline

UR - https://www.scopus.com/pages/publications/79952588931

U2 - 10.1007/978-1-59745-248-9_18

DO - 10.1007/978-1-59745-248-9_18

M3 - Journal article

C2 - 21125496

SN - 1064-3745

VL - 703

SP - 265

EP - 273

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -

Isolation of RNP granules

Abstract

Keywords

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