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PMID: 7682707 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Visualization of the interaction of a regulatory protein with RNA in vivo.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 90 ·No. 8 ·1993-04-15 ·Pages 3496-500

Bertrand E, Fromont-Racine M, Pictet R, Grange T

Abstract

We have adapted to RNA molecules the ligation-mediated polymerase chain reaction (LMPCR) procedure of genomic sequencing [Mueller, P. R. & Wold, B. (1989) Science 246, 780-786]. This new procedure, the reverse ligation-mediated PCR (RLPCR), is sufficiently sensitive to allow "in vivo" footprinting of minor RNA species. It is based on the ligation of an RNA linker of known sequence to every 5' end resulting from the cleavage of total cellular RNA. Target RNA molecules are specifically reverse-transcribed and the resulting products are amplified by PCR. The localization of the initial 5' ends is ultimately determined on a sequencing gel. To demonstrate the validity of this strategy, we have used RNase T1 treatment of permeabilized cells and RLPCR and have detected in vivo iron-depletion-dependent footprints on two iron-responsive elements of the transferrin receptor mRNA.

MeSH Terms
Base Sequence Carcinoma, Hepatocellular DNA, Neoplasm/genetics,isolation & purification Deferoxamine/pharmacology Gene Expression Regulation, Neoplastic Hemin/pharmacology Humans Liver Neoplasms Molecular Sequence Data Nucleic Acid Conformation Oligodeoxyribonucleotides/chemical synthesis Polymerase Chain Reaction/methods RNA, Messenger/genetics,isolation & purification,metabolism RNA, Neoplasm/genetics,isolation & purification,metabolism RNA-Binding Proteins/metabolism RNA-Directed DNA Polymerase/metabolism Receptors, Transferrin/genetics Ribonuclease T1/metabolism Tumor Cells, Cultured
Chemicals
DNA, Neoplasm Oligodeoxyribonucleotides RNA, Messenger RNA, Neoplasm RNA-Binding Proteins Receptors, Transferrin Hemin RNA-Directed DNA Polymerase Ribonuclease T1 Deferoxamine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bertrand E
Institut Jacques Monod, Centre National de la Recherche Scientifique, Université Paris, France.
Fromont-Racine M
Pictet R
Grange T
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.90.8.3496
Published
1993-04-15
Pages
3496-500
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC46327
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.90.8.3496 PMC Full Text View journal details
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