Comparing Proteomics and RISC Immunoprecipitations to Identify Targets of Epstein-Barr Viral miRNAs

Malika Kuzembayeva,Ya-Fang Chiu,Bill Sugden,Ashok Aiyar

doi:10.1371/journal.pone.0047409

Malika Kuzembayeva, Ya-Fang Chiu + Show 2 more

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https://doi.org/10.1371/journal.pone.0047409

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Journal: PloS one	Publication Date: Oct 16, 2012
Citations: 35	License type: CC BY 4.0

Affiliation: University of Wisconsin–Madison

Abstract

Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV’s life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV’s miRNAs.

Highlights

MiRNAs are small, noncoding RNAs of about 20–22 nucleotides in length that regulate gene expression at the posttranscriptional level
The Epstein-Barr virus (EBV)-negative Burkitt’s lymphoma cell line, BJAB, which was either transduced with a retrovirus that expresses BamHI A Rightward transcript (BART) miRNAs constitutively (BJAB BARTs) or an empty virus vector (BJAB Empty) was used to identify cellular proteins whose expression levels are regulated by EBV’s miRNAs. This approach provided as isogenic cell lines as available currently, where changes in protein expression could be attributed to the effects of regulation by BART miRNAs alone, with no other viral genes being present
We looked for any insights in comparing our two approaches we could glean from analyzing the candidates they uncovered using the program Probability of Interaction by Target Accessibility (PITA). 39UTRs of 35 candidates identified by 2-dimensional Differential Gel Electrophoresis (2D DIGE) and of 135 candidates identified by RNA Induced Silencing Complexes (RISCs) IPs, which were enriched at least at levels comparable to TOMM22 were screened for predicted binding sites of BART miRNAs

Summary

Introduction

MiRNAs are small, noncoding RNAs of about 20–22 nucleotides in length that regulate gene expression at the posttranscriptional level. They have been implicated as regulators of most cellular processes with abnormal expression of miRNAs reported in various diseases including cancer [1,2]. Regulation of gene expression by the miRNAs involves their binding to complimentary sequences in target mRNAs, and guiding the RNA Induced Silencing Complexes (RISCs) to those mRNAs, resulting in a combination of mRNA degradation and inhibition of translation. The latent phase of EBV’s life-cycle, a phase in which no viral progeny are produced, is characterized by the expression of different viral genes in the infected cell, but always include the miRNAs encoded by the BART transcript [6,9]

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