Molecular Basis for the Central Role of the DEAD-Box Helicase Dhh1 in the Formation and Function of Processing Bodies

Processing-bodies (P-bodies) contain the machinery that is involved in 5ʼ to 3ʼ mRNA degradation. This includes the DEAD box RNA helicase Dhh1 as well as the mRNA decapping enzyme Dcp2, proteins that interact with the decapping enzyme (Dcp1, Pat1, LSm1-7, Edc1, Edc3), the 3ʼ to 5ʼ exonuclease Xrn1 and translationally inactive mRNA. The Dhh1 enzyme is involved in RNP assembly, in translational repression and in the degradation of mRNA. The hot-spot function of Dhh1 in P-body formation is underscored by recent observations that show that the helicase undergoes spontaneous LLPS in vitro in the presence of RNA and ATP as well as by studies that show that the size of in vivo P-bodies is altered upon Dhh1 mutations.

Here, we aim at unraveling the atomic details of the network of intermolecular interactions between Dhh1, mRNA and other P-body components. To that end, we make use of an in vitro bottom up approach to assay LLPS of a mixture of purified and well defined P-body components. These experiments are complemented with high resolution NMR studies that identify residues that are at the center of the interaction interfaces.

Current State of Research

We have cloned and expressed most proteins and they behave well in biochemical and biophysical experiments. This now allows us to start mapping interactions and to perform high throughput in vitro LLPS assays