A proteomic study of TAR-RNA binding protein (TRBP)-associated factors
© Chi et al; licensee BioMed Central Ltd. 2011
Received: 27 January 2011
Accepted: 25 February 2011
Published: 25 February 2011
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.
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.
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.
MicroRNAs (miRNAs) are important regulators of cellular development, differentiation, proliferation, apoptosis, metabolism, and oncogenesis [1–4]. Cellular miRNAs have also been proposed to function in restricting virus infection of cells [5–11]. The biogenesis of miRNAs in eukaryotic cells has been reviewed extensively elsewhere [12, 13]. In brief, RNA polymerase II transcribed primary microRNA transcripts (pri-miRNA) are processed by Drosha in the nucleus to pre-miRNA precursors which are then processed by Dicer in the cytoplasm to mature miRNAs. In analyzing miRNA processing, it was noted that a cellular protein, TAR RNA-binding protein TRBP, binds directly to Dicer [14–17] and is important to the activity of miRNA-mediated RNA-silencing. Moreover, a TRBP-Ago2 composed RNA-induced silencing complex (RISC) has been shown to be needed for optimal miRNA-guided post-transcriptional silencing .
TRBP contains two double-stranded RNA binding domains (RBD)  and was originally characterized and cloned based on its high affinity binding to the HIV-1 small hairpin leader RNA, TAR [19–21]. Consistent with the notion of miRNA-mediated restriction of viral infection in mammalian cells, loss of TRBP activity through its sequestration by TAR RNA resulted in the enhanced replication of HIV-1 in human cells . To date, TRBP is considered to act inside cells through at least three different mechanisms: it can promote the translation of TAR RNA-containing viral RNAs ; it can directly bind and inhibit the interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase R (PKR) ; and as mentioned above, it can be a Dicer co-factor in the miRNA/siRNA RNAi pathway. To gain additional insight into TRBP function, we sought to identify human cellular factors that associate with TRBP. Here, using co-immunoprecipitation (co-IP) and protein-identification by mass spectrometry, we report proteins in a TRBP-proteome.
Functional categories of the TRBP-associated complex
ABCF1, EEF1A2, EIF2AK2, EIF2S1, PTBP1, RPL3, RPL4, RPL5, RPL6, RPL7, RPL9, RPL11, RPL15, RPL18, RPL21, RPL22, RPL24, RPL30, RPL32, RPL34, RPL35, RPL18A, RPL23A, RPL27A, RPL29 (includes EG:6159), RPL31 (includes EG:6160), RPL7A (includes EG:6130), RPS2, RPS5, RPS6, RPS9, RPS10, RPS11, RPS17 (includes EG:6218), RPS3A
RNA Post-Transcriptional Modification
DDX17, DDX54, EFTUD2, HNRPAB, HNRPC, HNRPUL1, PABPC1, PRPF8, PTBP1, SFRS1, SNRPD1, SNRPD3, SYNCRIP, U2AF1
Cellular Growth and Proliferation
AKAP13, ATP5B, ATP5F1, CCT2, DBN1, DCD, DNAJA3, EIF2AK2, GLTSCR2, HMGA1, HNRPA1, HNRPAB, HNRPC, HNRPF, HNRPM, PHB (includes EG:5245), PPP1R9B, PRPF8, PRPF19, RAP1B, RPS3A, RSL1D1, SART1, SFRS1, TOP1, XRN2
BASP1, BTF3, CDC5L, CHD4, CSDA, DDX5, DEK, EIF2S1, GTF3C1, GTF3C3, GTF3C4, HMGA1, HNRPAB, IGHMBP2, PARP1, PHB (includes EG:5245), RBM39, RPL6, SOX21, TARBP2, TOP1, TOP2A
DDX5, DIDO1, DNAJA3, EEF1A2, EIF2AK2, EIF2S1, GNL3, HIST1H1C, HMGA1, HNRPA1, HNRPC, HSPA1B, PARP1, PHB (includes EG:5245), PPP1R9B, PRPF19, RBBP4, RPLP0 (includes EG:6175), RPS3A, SLC2A1, TOP1, TOP2A
DNA Replication, Recombination, and Repair
CDC5L, HMGA1, HSPA1B, IGHMBP2, PARP1, PRPF19, TOP1, TOP2A
CDC5L, EIF2AK2, GNL3, HMGA1, NOL1, PARP1, PES1, PHB (includes EG:5245), PPP1R9B, PRMT5, RPL5, RPL11, RPL23, TOP2A
RPL5, RPL11, RPL23, AKAP13, ASPH, DNAJA3, HMGA1, HSPA1B, PES1, PRMT5, RPS3A
Skeletal and Muscular Disorders
RPL5, RPL11, RPL23
AKAP13, ASPH, DNAJA3, HMGA1, HSPA1B, PES1, PRMT5, RPS3A
Cellular Function and Maintenance/Connective Tissue Development and Function
AKAP13, EIF2AK2, HMGA1, PARP1, SFRS1
Although previous reports implicated TRBP as active primarily in the cytoplasm, our mass spectrometry results (Additional file 2. Supplementary Materials and Methods) identified several TRBP-interacting nuclear factors which are known to participate in processes including RNA splicing, cellular growth and proliferation and gene transcription (Figure 2, Table 1; and Additional file 1, Table S1). For example, EFTUD2, HNRPC, PRPF8, PTBP1, SFRS1, SYNCRIP, SNRPD1, SNRPD3 and U2AF1 are components of the small nuclear ribonucleoprotein (snRNPs) that are involved in splicing of pre-mRNAs. Related to gene transcription, TRBP-associated proteins include those found to be active in RNA polymerase II- and III-dependent transcription (Additional file 1, Table S1). Additionally, a TRBP-associated factor, Sox21, is a HMG-box transcription factor that acts in a complex array of repressive and activating transcriptional processes in embryonic stem cells . The transcription co-activator HMGA1 was also identified in our proteomic data (Table 1). Whether TRBP participates in the determination of pluripotency through Sox21 requires further study.
Recently, TRBP truncating mutations were found in human cancers with microsatellite instability . Evidence exists which supports the notion that impaired miRNA processing and destabilization of the DICER1 protein are correlated with tumorigenesis . Because TRBP is known to interact with Dicer and to be involved in mRNA processing, how TRBP and TRBP-interacting proteins contribute to oncogenesis merits future investigation. The current TRBP proteome provides a starting point for initiating those explorations. Our study describes the TRBP-proteome in HeLa cells. Although TRBP was originally identified as the HIV-1 TAR RNA-binding protein, its more recently characterized function in microRNA biogenesis and RISC-activity is widely conserved in all cell types (e.g. epithelial, hematopoietic, mesenchymal cells etc...). Thus, a HeLa-like TRBP-proteome is likely to be found in all types of human cells. We realize that systematic functional validation of the currently identified TRBP proteome will be necessary in order to clarify the biological relevance of each factor. Indeed, some of the TRBP-interacting proteins are being currently characterized.
TAR-RNA binding protein 2
RNA-induced silencing complex
trans-activation responsive element.
Work in the KTJ laboratory was supported in part by NIAID/NIH intramural funds and by the Intramural AIDS Targeted Anti-viral Program (IATAP).
- Garzon R, Fabbri M, Cimmino A, Calin GA, Croce CM: MicroRNA expression and function in cancer. Trends Mol Med. 2006, 12: 580-587. 10.1016/j.molmed.2006.10.006View ArticlePubMed
- Svoboda P, Flemr M: The role of miRNAs and endogenous siRNAs in maternal-to-zygotic reprogramming and the establishment of pluripotency. EMBO Rep. 2010, 11: 590-597. 10.1038/embor.2010.102PubMed CentralView ArticlePubMed
- Lewis MA, Steel KP: MicroRNAs in mouse development and disease. Semin Cell Dev Biol. 2010, 21: 774-780. 10.1016/j.semcdb.2010.02.004PubMed CentralView ArticlePubMed
- Yeung ML, Bennasser Y, Jeang KT: miRNAs in the biology of cancers and viral infections. Curr Med Chem. 2007, 14: 191-197. 10.2174/092986707779313417View ArticlePubMed
- Berkhout B, Jeang KT: RISCy business: MicroRNAs, pathogenesis, and viruses. J Biol Chem. 2007, 282: 26641-26645. 10.1074/jbc.R700023200View ArticlePubMed
- Watanabe Y, Kishi A, Yachie N, Kanai A, Tomita M: Computational analysis of microRNA-mediated antiviral defense in humans. FEBS Lett. 2007, 581: 4603-4610. 10.1016/j.febslet.2007.08.049View ArticlePubMed
- Yeung ML, Benkirane M, Jeang KT: Small non-coding RNAs, mammalian cells, and viruses: regulatory interactions?. Retrovirology. 2007, 4: 74. 10.1186/1742-4690-4-74PubMed CentralView ArticlePubMed
- Grassmann R, Jeang KT: The roles of microRNAs in mammalian virus infection. Biochim Biophys Acta. 2008, 1779: 706-711.PubMed CentralView ArticlePubMed
- Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K: Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science. 2007, 315: 1579-1582. 10.1126/science.1136319View ArticlePubMed
- Chable-Bessia C, Meziane O, Latreille D, Triboulet R, Zamborlini A, Wagschal A: Suppression of HIV-1 replication by microRNA effectors. Retrovirology. 2009, 6: 26. 10.1186/1742-4690-6-26PubMed CentralView ArticlePubMed
- Strebel K, Luban J, Jeang KT: Human cellular restriction factors that target HIV-1 replication. BMC Med. 2009, 7: 48. 10.1186/1741-7015-7-48PubMed CentralView ArticlePubMed
- Jaskiewicz L, Filipowicz W: Role of Dicer in posttranscriptional RNA silencing. Curr Top Microbiol Immunol. 2008, 320: 77-97. full_textPubMed
- Ambros V: The functions of animal microRNAs. Nature. 2004, 431: 350-355. 10.1038/nature02871View ArticlePubMed
- Haase AD, Jaskiewicz L, Zhang H, Laine S, Sack R, Gatignol A: TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 2005, 6: 961-967. 10.1038/sj.embor.7400509PubMed CentralView ArticlePubMed
- Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K: TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature. 2005, 436: 740-744. 10.1038/nature03868PubMed CentralView ArticlePubMed
- Kok KH, Ng MH, Ching YP, Jin DY: Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem. 2007, 282: 17649-17657. 10.1074/jbc.M611768200View ArticlePubMed
- Daniels SM, Melendez-Pena CE, Scarborough RJ, Daher A, Christensen HS, El FM: Characterization of the TRBP domain required for dicer interaction and function in RNA interference. BMC Mol Biol. 2009, 10: 38. 10.1186/1471-2199-10-38PubMed CentralView ArticlePubMed
- Watashi K, Yeung ML, Starost MF, Hosmane RS, Jeang KT: Identification of small molecules that suppress microRNA function and reverse tumorigenesis. J Biol Chem. 2010, 285: 24707-24716. 10.1074/jbc.M109.062976PubMed CentralView ArticlePubMed
- Gatignol A, Buckler-White A, Berkhout B, Jeang KT: Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991, 251: 1597-1600. 10.1126/science.2011739View ArticlePubMed
- Gatignol A, Buckler C, Jeang KT: Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen. Mol Cell Biol. 1993, 13: 2193-2202.PubMed CentralView ArticlePubMed
- Duarte M, Graham K, Daher A, Battisti PL, Bannwarth S, Segeral E: Characterization of TRBP1 and TRBP2. Stable stem-loop structure at the 5' end of TRBP2 mRNA resembles HIV-1 TAR and is not found in its processed pseudogene. J Biomed Sci. 2000, 7: 494-506. 10.1007/BF02253365View ArticlePubMed
- Bennasser Y, Yeung ML, Jeang KT: HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP. J Biol Chem. 2006, 281: 27674-27678. 10.1074/jbc.C600072200View ArticlePubMed
- Dorin D, Bonnet MC, Bannwarth S, Gatignol A, Meurs EF, Vaquero C: The TAR RNA-binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR. J Biol Chem. 2003, 278: 4440-4448. 10.1074/jbc.M208954200View ArticlePubMed
- Benkirane M, Neuveut C, Chun RF, Smith SM, Samuel CE, Gatignol A: Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR. EMBO J. 1997, 16: 611-624. 10.1093/emboj/16.3.611PubMed CentralView ArticlePubMed
- Chendrimada TP, Finn KJ, Ji X, Baillat D, Gregory RI, Liebhaber SA: MicroRNA silencing through RISC recruitment of eIF6. Nature. 2007, 447: 823-828. 10.1038/nature05841View ArticlePubMed
- Wilson DN, Nierhaus KH: Ribosomal proteins in the spotlight. Crit Rev Biochem Mol Biol. 2005, 40: 243-267. 10.1080/10409230500256523View ArticlePubMed
- Chakravarthy H, Ormsbee BD, Mallanna SK, Rizzino A: Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery. FASEB J. 2011, 25: 206-218. 10.1096/fj.10-166926PubMed CentralView ArticlePubMed
- Melo SA, Ropero S, Moutinho C, Aaltonen LA, Yamamoto H, Calin GA: A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function. Nat Genet. 2009, 41: 365-370. 10.1038/ng.317PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. 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.