Identification and analysis of dicer associated proteins in human cells

Abstract

Dicer is a multidomain RNase III like enzyme involved in the initial steps of RNA interference (RNAi) and microRNA (miRNA) pathways, two mechanisms of posttranscriptional gene silencing conserved throughout most eukaryotic species. Dicer has been shown to be necessary and sufficient to cut long dsRNA and miRNA precursors into small (21-25 nt) RNAs. In metazoa, the small RNA products of Dicer are further incorporated into a multiprotein RNA induced silencing complex (RISC), which target mRNAs in a sequence specific manner to induce mRNA cleavage (guided by siRNAs) or inhibition of translation (guided by miRNAs). We aimed to identify proteins interacting with human Dicer. Specific anti- Dicer antibodies were used to immunoprecipitate Dicer from HEK293 and HeLa cells and co-immunoprecipitating proteins were analyzed by mass spectrometry. Proteins identified to specifically co-purify with Dicer fall into different categories: (1) known components of RNA silencing pathways, such as Argonaute proteins; (2) translation factors; (3) RNA helicases; (4) RNA binding proteins, and others. Among the proteins characterized by mass spectrometry we identified TRBP [HIV-1 transactivating response (TAR) RNA-binding protein], as a protein containing three dsRNA binding domains (dsRBD). We found that this protein interacts with human Dicer in vivo and in vitro, and that this interaction involves Dicer’s N-terminal helicase domain and TRBP’s third dsRBD. TRBP has previously been assigned several different functions, including inhibition of the interferon (IFN)-induced dsRNA-regulated protein kinase PKR, modulation of HIV-1 gene expression through its association with TAR, and control of cell growth. To analyze TRBP’s function in miRNA and siRNA pathways, we generated stable TRBP knock down cell lines. Using a Luciferase reporter system we showed that TRBP is necessary for efficient silencing mediated by endogenous miRNAs. Dicer cleavage activity tested in vitro using a pre-miRNA substrate was impaired in TRBP knock down extracts. However, endogenous miRNA levels were not significantly changed upon knock down of TRBP. We also found that RNAi against an endogenous gene, induced by transfection of siRNAs, was impaired in TRBP knock down cell lines. Taken together our observations show that TRBP interacts with Dicer, and suggest that, besides its impact on Dicer cleavage activity in vitro, TRBP functions mainly downstream of Dicer cleavage in miRNA and siRNA pathways. Our data provide support to the notion that large RNaseIII-type Drosha and Dicer nucleases work in conjunction with small dsRBD protein partners. They also raise the possibility of a cross talk between RNA silencing and the IFN/PKR pathways in normal and virus-infected cells. To further characterize the Dicer/TRBP complex, we generated anti-TRBP antibodies, and analyzed TRBP immunoprecipitates by mass spectrometry. Data obtained from peptide sequencing of Dicer and TRBP immunopurifications were compared, and proteins identified in both preparations, among them the E3 Ubiquitin ligase Ro52, were considered for further analyses as likely components of a Dicer/TRBP complex. Ro52, also known as Sjoegren Syndrome Antigen 1 (SSA-1) or Tripartite motif protein 21 (TRIM21) was first identified as one of the major autoantigens in Sjoegren Syndrome and Systemic Lupus Erythematosus (SLE), two severe human autoimmune diseases. Recently, it was demonstrated that Ro52 can act as a RING dependent E3 Ubiquitin ligase, and the E3 ligase activity of Ro52 was shown to be required for S-phase progression in mammalian cells. We found that Ro52 associates with the Dicer/TRBP complex, and interacts with TRBP in vitro. The presence of an E3 Ubiquitin ligase in the Dicer/TRBP complex raised the possibility that components of the RNA silencing complexes are post-translationally modified by Ubiquitin. Our data indicate that TRBP can be covalently tagged by Ubiquitin. It appears that TRBP is monoubiquitinated, rather than polyubiquitinated and degraded by the proteasome. Future work will be required to establish the function of ubiquitination of TRBP, and the role of Ro52 in RNA silencing pathways.