RNase R is a conserved exoribonuclease in the RNase II family that primarily participates in RNA decay in all kingdoms of life. RNase R degrades duplex RNA with a 3′ overhang, suggesting that it has RNA unwinding activity in addition to its 3′-to-5′ exoribonuclease activity. However, how RNase R coordinates RNA binding with unwinding to degrade RNA remains elusive. Here, we report the crystal structure of a truncated form of Escherichia coli RNase R (residues 87–725) at a resolution of 1.85 Å. Structural comparisons with other RNase II family proteins reveal two open RNA-binding channels in RNase R and suggest a tri-helix ‘wedge’ region in the RNB domain that may induce RNA unwinding. We constructed two tri-helix wedge mutants and they indeed lost their RNA unwinding but not RNA binding or degrading activities. Our results suggest that the duplex RNA with an overhang is bound in the two RNA-binding channels in RNase R. The 3′ overhang is threaded into the active site and the duplex RNA is unwound upon reaching the wedge region during RNA degradation. Thus, RNase R is a proficient enzyme, capable of concurrently binding, unwinding and degrading structured RNA in a highly processive manner during RNA decay.
Aberrant expression, dysfunction and particularly aggregation of a group of RNA-binding proteins, including TDP-43, FUS and RBM45, are associated with neurological disorders. These three disease-linked RNA-binding proteins all contain at least one RNA recognition motif (RRM). However, it is not clear if these RRMs contribute to their aggregation-prone character. Here, we compare the biophysical and fibril formation properties of five RRMs from disease-linked RNA-binding proteins and five RRMs from non-disease-associated proteins to determine if disease-linked RRMs share specific features making them prone to self-assembly. We found that most of the disease-linked RRMs exhibit reversible thermal unfolding and refolding, and have a slightly lower average thermal melting point compared to that of normal RRMs. The full domain of TDP-43 RRM1 and FUS RRM, as well as the β-peptides from these two RRMs, could self-assemble into fibril-like aggregates which are amyloids of parallel β-sheets as verified by X-ray diffraction and FT-IR spectroscopy. Our results suggest that some disease-linked RRMs indeed play important roles in amyloid formation and shed light on why RNA-binding proteins with RRMs are frequently identified in the cellular inclusions of neurodegenerative diseases.
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