Investigation of protein-RNA interactions of the TAR-RNA-binding protein (TRBP)

Open Access
Acevedo, Roderico
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
February 26, 2016
Committee Members:
  • Scott A Showalter, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Member
  • Craig Eugene Cameron, Committee Member
  • Amie Kathleen Boal, Committee Member
  • Ken S Feldman, Special Member
  • Biophysics
  • Binding
  • TRBP
  • miRNA
  • RNAi
Post-transcriptional regulation of gene expression is dependent on multiple RNA silencing pathways, termed generally as RNA interference (RNAi). Silencing occurs through use of single-stranded RNAs, produced as double-stranded precursors, in two main modes: microRNA (miRNA) in multicellular organisms and small interfering RNA (siRNA) primarily in unicellular organisms. In order to prevent the degradation of these important RNAs, double-stranded RNA binding proteins (dsRBP) play a pivotal role in the high fidelity trafficking of RNA in the cell. Here we focus on the human immunodeficiency virus trans-activating protein response RNA binding protein (TRBP), a cofactor of the RNase III enzyme Dicer. We hypothesize that the intramolecular interactions between the dsRBDs in TRBP lead to high affinity double-stranded RNA (dsRNA) binding and ultimately, to higher specificity for the subset of dsRNA that are targeted for Dicer processing. TRBP contains 3 double-stranded RNA binding domains (dsRBDs). Here, we focus on the first two dsRBDs, which have been shown to bind dsRNA. By monitoring the binding affinity of the first two dsRBDs of TRBP individually (TRBP-dsRBD1 and TRBP-dsRBD2) and in tandem (referred to as TRBP-ΔC) with respects to various lengths of Watson-Crick duplex (W-C) RNA, we saw that TRBP-ΔC bound 10-fold tighter than the individual dsRBDs at every given length. This led us to hypothesize that the observed binding affinity in TRBP-ΔC was due to the flexible linker region between TRBP-dsRBD1 and TRBP-dsRBD2. A subsequent linker length deletion studies showed that the linker length between dsRBDs correlates with binding affinity of the tandem complex. This result was bolstered by our circular dichroism results that showed there was no difference in total number of dsRBDs binding to a given length of RNA between the individual dsRBDs and that of TRBP-ΔC. The combined results suggest that multi-dsRBD proteins, like TRBP, benefit from some form of intra-chain interactions. Further inspection of TRBP-ΔC’s binding thermodynamics using isothermal titration calorimetry revealed that changing the linker length between dsRBDs modulates TRBP-ΔC binding affinity. Jacobson-Stockmayer analysis of the linker length data suggested that TRBP-ΔC may exhibit negative intra-chain cooperativity (i.e., between dsRBDs within the same molecule of TRBP-ΔC) as the linker region contributes unfavorably to the Gibbs free energy of binding. This result was corroborated independently through a re-examination of the EMSA data, where application of a biophysically relevant model also suggested a negative intra-chain cooperativity of TRBP-ΔC binding. However, this same model demonstrated that the negative intra-chain cooperativity is overwhelmed by the positive inter-molecular cooperativity (i.e., between different molecules of TRBP-ΔC binding to the same RNA). More importantly, cross-analysis between the W-C RNA binding study and that of RNAs which contain helical imperfections demonstrated that TRBP-ΔC is able to distinguish between RNAs with helical imperfections, such as miRNAs, from those without imperfections, such as siRNAs. These results highlight how TRBP can selectively bind to precursor miRNA, over other cellular RNAs, and deliver it to Dicer for downstream processing.