Biophysical Studies of Asymmetric Homodimerisation of the microRNA Biogenesis Cofactors PACT and TRBP

Processing of precursor microRNAs by Dicer is a key step in microRNA biogenesis. This process is assisted by the homologous proteins PACT and TRBP, which bind to the helicase domain of Dicer. The mechanism by which they assist microRNA biogenesis is poorly understood, but could include facilitating substrate positioning, assisting Argonaute loading, or discriminating between different classes of pre-miRNA.
PACT also regulates innate immune pathways that respond to viral double-stranded RNA, including via the kinase PKR. Mutations in PACT lead to early onset dystonia in humans, while depletion of PACT in mice results in both growth and fertility defects: both have been linked to inappropriate or altered activation of PKR. Homodimerisation of PACT via its C-terminal domain (PACT-D3) is thought to be necessary for it to induce PKR activation.
Homodimerisation of wild-type and mutant constructs of PACT-D3 was assayed using biophysical techniques. SEC-MALLS and analytical ultracentrifugation data demonstrate that PACT-D3 homodimerises via a different mechanism to a previously reported dsRBD homodimer, dsRBD-5 of Staufen1. Instead, NMR analyses show that PACT-D3 forms an asymmetric homodimer similar to that observed in the Drosophila melanogaster homologue Loquacious. Dimerisation could be abolished by the L273R mutation, while phospho-mimic mutations did not appear to significantly affect dimerisation. TRBP domain 3 also forms asymmetric dimers, but with weaker affinity due to sequence differences in its C- terminal -helix. Asymmetry is caused by a register shift between intermolecular parallel beta-strands, but the functional significance of asymmetric homodimerisation remains unclear.
The data presented in this thesis supports a model in which the homodimerisation interface of PACT-D3 overlaps with the surface that binds to Dicer, and suggests that PACT homodimerisation and the formation of a Dicer-PACT complex are incompatible.