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.
The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human DEDDh exonucleases can serve as antiviral drug targets due to their critical roles in virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition mechanism by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications.
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