Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unk...

Full description

Bibliographic Details
Main Authors:	Hu, Yi, Yin, Kun-Lun, Ma, Xu, Xia, Hong-Fei
Format:	Online
Language:	English
Published:	Public Library of Science 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118908/

id	pubmed-4118908
recordtype	oai_dc
spelling	pubmed-41189082014-08-04 Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA Hu, Yi Yin, Kun-Lun Ma, Xu Xia, Hong-Fei Research Article MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unknown, miRNAs have been implicated in diverse biological processes, including both normal and pathological states. As a single stranded mRNA can be directly targeted by multiple miRNAs, and as the target sites may exist in the 3′-untranslated region (UTR), 5′-UTR, or the coding regions, it is essential to develop an effective method to identify the full-scale miRNA regulatory pattern of each particular gene. In this study, we employed a biochemical approach to identify the miRNA profiles that regulate the expression of embryonic ectoderm development (EED) protein by using anti-PABPC1 ribonucleoprotein (RNP) co-immunoprecipitation (Co-IP). The full length EED mRNA was subcloned into an expression vector and transiently transfected into a Flag-PABPC1 stable expression cell line. Subsequent to cross-linking and an anti-Flag Co-IP, the miRNAs that directly targeted EED were identified. We found that the best time point to distinguish the positive miRNAs from the background was 18 hours after the plasmid transfection. As expected, the miRNAs that directly target EED were found to interact with EED mRNA through the miRNA-induced silencing complex (miRISC). Meanwhile, the EED mRNA was bound by Flag-PABPC1. This method depends on the integrity of the miRISC complex and achieves greater efficiency when ultraviolet irradiation is used for the process of cross-linking. By using anti-PABPC1 RIP, we identified EED to be a new target gene of miR-16; a finding further confirmed using a dual-luciferase assay. In summary, our data indicate that anti-PABPC1 RIP is a validated and direct biochemical method to provide data about specific miRNA-mRNA interactions, as well as global miRNA patterns regulating the mRNAs. Public Library of Science 2014-08-01 /pmc/articles/PMC4118908/ /pubmed/25084349 http://dx.doi.org/10.1371/journal.pone.0103695 Text en © 2014 Hu et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Hu, Yi Yin, Kun-Lun Ma, Xu Xia, Hong-Fei
spellingShingle	Hu, Yi Yin, Kun-Lun Ma, Xu Xia, Hong-Fei Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA
author_facet	Hu, Yi Yin, Kun-Lun Ma, Xu Xia, Hong-Fei
author_sort	Hu, Yi
title	Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA
title_short	Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA
title_full	Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA
title_fullStr	Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA
title_full_unstemmed	Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA
title_sort	anti-pabpc1 co-immunoprecipitation for examining the mirnas directly targeting the 3′-utr of eed mrna
description	MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unknown, miRNAs have been implicated in diverse biological processes, including both normal and pathological states. As a single stranded mRNA can be directly targeted by multiple miRNAs, and as the target sites may exist in the 3′-untranslated region (UTR), 5′-UTR, or the coding regions, it is essential to develop an effective method to identify the full-scale miRNA regulatory pattern of each particular gene. In this study, we employed a biochemical approach to identify the miRNA profiles that regulate the expression of embryonic ectoderm development (EED) protein by using anti-PABPC1 ribonucleoprotein (RNP) co-immunoprecipitation (Co-IP). The full length EED mRNA was subcloned into an expression vector and transiently transfected into a Flag-PABPC1 stable expression cell line. Subsequent to cross-linking and an anti-Flag Co-IP, the miRNAs that directly targeted EED were identified. We found that the best time point to distinguish the positive miRNAs from the background was 18 hours after the plasmid transfection. As expected, the miRNAs that directly target EED were found to interact with EED mRNA through the miRNA-induced silencing complex (miRISC). Meanwhile, the EED mRNA was bound by Flag-PABPC1. This method depends on the integrity of the miRISC complex and achieves greater efficiency when ultraviolet irradiation is used for the process of cross-linking. By using anti-PABPC1 RIP, we identified EED to be a new target gene of miR-16; a finding further confirmed using a dual-luciferase assay. In summary, our data indicate that anti-PABPC1 RIP is a validated and direct biochemical method to provide data about specific miRNA-mRNA interactions, as well as global miRNA patterns regulating the mRNAs.
publisher	Public Library of Science
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118908/
_version_	1613120028228452352

Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA

Similar Items