Poster abstracts

Poster number 75 submitted by Jeff Levengood

Strategies for utilizing NMR to gain insights into an intrinsically disordered domain of hnRNP A1

Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Julien Roche (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
The hnRNP A1 protein is involved in numerous processes of nascent RNA transcripts. These roles include, but are not limited to, translational control, splicing regulation, and mRNA stabilization. The protein performs its functions by first binding the RNA at a high affinity site before recruiting other proteins to aid in its RNA processing activities.

hnRNP A1 is composed of three domains, two structurally identical RRMs that form the nucleic acid binding protein UP1, and a disordered, glycine rich C-terminal domain (G-CTD). As the more structured domain, UP1 has been studied extensively. Its RNA binding properties have been examined through numerous biochemical and biophysical methods. Its structure, both free and bound, has been determined through NMR, X-ray crystallography, and small-angle X-ray scattering (SAXS).

In contrast to UP1, the G-CTD has not been very well characterized. However, its roles in hnRNP A1 activity requires that it be studied with just as much detail as UP1. The G-CTD is responsible for assembling the multi-protein complexes the protein utilizes to carry out its various functions. It has been shown to be a low complexity sequence domain (LCD) capable of mediating liquid-liquid phase separation (LLPS). This partitioning leads to the formation of stress granules, cytosolic bodies which are believed to be formed by mRNAs stalled in translation.

Experiments in high-pressure NMR on full length hnRNP A1 have revealed an interesting property of the G-CTD; its HSQC peaks disappear at high pressure. At 2,500 bar, the hnRNP A1 peaks that are part of the UP1 domain overlap with peaks from the isolated UP1 domain at identical pressure. The two spectra are completely identical as all peaks from the G-CTD are not present. The reason for this is currently unknown. Further experiments with the isolated G-CTD domain will be performed in order to gain further insight into this phenomenon.

Keywords: RRM, Stress Granules