TRBP contains two double-stranded RNA binding domains (RBD) [17] 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 [22]. 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 [23]; it can directly bind and inhibit the interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase R (PKR) [24]; 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.

Figure ​Figure1A1A shows the Coomassie blue stained profile of proteins that co-IP with TRBP. After treating with RNase A, the majority (> 80%) of the co-IP products were reduced in amounts, suggesting that many TRBP-interacting partners in the co-IP are indirectly linked via RNA-association (silver stain, Figure ​Figure1B).1B). Next, we compared "Mock" to "TRBP" samples based on silver staining; and we excised the corresponding Coomassie blue stained protein bands that were enhanced in the TRBP sample. These bands were then analyzed by LC-MS/MS spectrometry (Figure ​(Figure1A).1A). The corresponding gel bands from the "Mock" sample were also excised and analyzed in parallel. By subtracting the "Mock" protein identification data from the "TRBP" protein identification data, 160 "TRBP-associated" factors were identified (Additional file 1, Table S1). The functional relevance (according to the p value computed by the Ingenuity Systems algorithm using right-tailed Fisher Exact Test) of each category of protein factors to established pathways is diagrammed in Figure ​Figure2,2, and the protein identities present in each functional category are listed in Table ​Table1.1. Similar to a previous report [25], our results show that TRBP is significantly associated with factors in the protein synthesis machinery that is constituted by the 80S ribosome [20 out of 32 proteins (RPSs; ribosomal protein, small subunits) of the 40S ribosomal subunits, and 24 out of 46 proteins (RPLs; ribosomal protein, large subunits) of the 60S ribosomal subunit were identified] [26]. Also identified were several translation elongation factors (e.g. ABCF1, EEF1A2, EIF2AK2 and EIF2S1) (Table ​(Table11).

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 ​(Figure2,2, Table ​Table1;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 [27]. The transcription co-activator HMGA1 was also identified in our proteomic data (Table ​(Table1).1). Whether TRBP participates in the determination of pluripotency through Sox21 requires further study.