A proteomic study of TAR-RNA binding protein (TRBP)-associated factors

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Abstract

Background

The human TAR RNA-binding protein, TRBP, was first identified and cloned based on its high affinity binding to the small hairpin trans-activation responsive (TAR) RNA of HIV-1. TRBP has more recently been found to be a constituent of the RNA-induced silencing complex (RISC) serving as a Dicer co-factor in the processing of the ~70 nucleotide pre-microRNAs(miRNAs) to 21-25 nucleotide mature miRNAs.

Findings

Using co-immunoprecipitation and protein-identification by mass spectrometry, we characterized intracellular proteins that complex with TRBP. These interacting proteins include those that have been described to act in protein synthesis, RNA modifications and processing, DNA transcription, and cell proliferation.

Conclusions

Our findings provide a proteome of factors that may cooperate with TRBP in activities such as miRNA processing and in RNA interference by the RISC complex.

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Most cited references 23

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MicroRNA silencing through RISC recruitment of eIF6.

Melissa A. Pasquinelli, Ramin Shiekhattar, Stephen Liebhaber … (2007)

MicroRNAs (miRNAs) are a class of small RNAs that act post-transcriptionally to regulate messenger RNA stability and translation. To elucidate how miRNAs mediate their repressive effects, we performed biochemical and functional assays to identify new factors in the miRNA pathway. Here we show that human RISC (RNA-induced silencing complex) associates with a multiprotein complex containing MOV10--which is the homologue of Drosophila translational repressor Armitage--and proteins of the 60S ribosome subunit. Notably, this complex contains the anti-association factor eIF6 (also called ITGB4BP or p27BBP), a ribosome inhibitory protein known to prevent productive assembly of the 80S ribosome. Depletion of eIF6 in human cells specifically abrogates miRNA-mediated regulation of target protein and mRNA levels. Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of the endogenous LIN-14 and LIN-28 target protein and mRNA levels. These results uncover an evolutionarily conserved function of the ribosome anti-association factor eIF6 in miRNA-mediated post-transcriptional silencing.

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A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function.

Luiz A S Melo, Catia Moutinho, L Aaltonen … (2009)

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNA (mRNA) transcripts. Recently, a miRNA expression profile of human tumors has been characterized by an overall miRNA downregulation. Explanations for this observation include a failure of miRNA post-transcriptional regulation, transcriptional silencing associated with hypermethylation of CpG island promoters and miRNA transcriptional repression by oncogenic factors. Another possibility is that the enzymes and cofactors involved in miRNA processing pathways may themselves be targets of genetic disruption, further enhancing cellular transformation. However, no loss-of-function genetic alterations in the genes encoding these proteins have been reported. Here we have identified truncating mutations in TARBP2 (TAR RNA-binding protein 2), encoding an integral component of a DICER1-containing complex, in sporadic and hereditary carcinomas with microsatellite instability. The presence of TARBP2 frameshift mutations causes diminished TRBP protein expression and a defect in the processing of miRNAs. The reintroduction of TRBP in the deficient cells restores the efficient production of miRNAs and inhibits tumor growth. Most important, the TRBP impairment is associated with a destabilization of the DICER1 protein. These results provide, for a subset of human tumors, an explanation for the observed defects in the expression of mature miRNAs.

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Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA.

Kin Hang Kok, Ming-Him James Ng, Yick-Pang Ching … (2007)

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.

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Author and article information

Journal

Journal ID (nlm-ta): Cell Biosci

Title: Cell & Bioscience

Publisher: BioMed Central

ISSN (Electronic): 2045-3701

Publication date Collection: 2011

Publication date (Electronic): 25 February 2011

Volume: 1

Page: 9

Affiliations

[1 ]Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan

[2 ]Department of Microbiology and Molecular Cell Biology, The Leroy T. Canoles Jr. Cancer Research Center,, Eastern Virginia Medical School, Norfolk, Virginia, USA

[3 ]Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Article

Publisher ID: 2045-3701-1-9

DOI: 10.1186/2045-3701-1-9

PMC ID: 3125213

PubMed ID: 21711701

SO-VID: 96718eee-bb8e-40b2-838b-84b9389fe245

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A proteomic study of TAR-RNA binding protein (TRBP)-associated factors

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Abstract

Background

Findings

Conclusions

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Claudins

Most cited references 23

MicroRNA silencing through RISC recruitment of eIF6.

A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function.

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA.

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