Structural and Biochemical Studies of the Novel Hexameric Endoribonuclease YicC

Abstract

The decay of mRNA is an essential process to bacteria. The newly identified E. coli protein YicC is a founding member of the UPF0701 family, and biochemical studies indicated that it is an RNase involved in mRNA degradation. However, its biochemical properties and catalytic mechanism are poorly understood. Here, we report the crystal structure of YicC, which shows an extended shape consisting of modular domains. While the backbone trace of the monomer forms a unique, nearly closed loop, the three monomers present in the asymmetric unit make a "shoulder-byshoulder" trimer. In vitro RNA cleavage assays indicated that this endoribonuclease mainly recognizes the consensus GUG motif, with a preference for an extended CGUG sequence. Additionally, the active enzyme exists as a hexamer in solution and assumes a funnel shape. Structural analysis indicated that the hexamer interface is mainly formed by the hexamerization domain consisting of D71− D124 and that the disruption of the oligomeric form greatly diminished the enzymatic activity. By studying the surface charge potential and the sequence conservation, we identified a series of residues that play critical functional roles, which helps to reveal the catalytic mechanism of this divalent metal-ion-dependent RNase. Last but not least, we discovered that the catalytic domain of YicC did not share similarity with any known nuclease fold, suggesting that the enzyme adopts a novel fold to perform its catalysis and in vivo functions. In summary, our investigations into YicC provide an in-depth understanding of the functions of the UPF0701 protein family and the DUF1732 domain in general.

Structural insights into RNA cleavage by a novel family of bacterial RNases

“…As we did here, Huang et al 9 also identified several arginine residues that contribute to RNA binding, including R30 and R280. Using a set of related RNA sequences as substrates, these authors reported that YicC recognizes primarily a GUG sequence.…”

“…While we were preparing this manuscript, Huang et al . published their findings on the structural and biochemical characterization of E. coli YicC 9 . These authors determined a low resolution YicC crystal structure (4.05 Å), and confirmed that the protein forms a hexamer.…”

“…The C-terminal domain is characterized as "DUF1732," i.e., a domain of unknown function without a reliable annotation in the Pfam database 7 , and it is in the top 20 of DUFs that are present in 500 or more species 8 . While we were preparing this manuscript, Huang et al published their ndings on the structural and biochemical characterization of E. coli YicC 9 . These authors determined a low resolution YicC crystal structure (4.05 Å), and con rmed that the protein forms a hexamer.…”