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

Abstract

Processing of RNA is a key regulatory mechanism for all living systems. We recently discovered a novel family of endoribonucleases that is conserved across all bacteria. Here, using crystallography, cryo-EM microscopy, biochemical, biophysical, and mass spectrometry techniques, we are able to shed light on a novel RNA cleavage mechanism in bacteria. We show that YicC, the prototypical member of this family, forms a hexameric channel that closes down on a 26-mer RNA substrate, and find that it cleaves across an RNA hairpin to generate several short fragments.

Figure 1. Crystal structure of apo YicC.

a, side view of YicC hexameric complex, with regions of the protein indicated. b, end view of the complex, focusing on the hexameric cap. The structures are colored according to chain.

Figure 2. Cryo-EM structure of YicC bound to substrate RNA.

a, electron density from cryo-EM structure, colored by chains. The map is shown in the same orientation as the apo structure in Fig. 1a. b, sectional view of overall structure, illustrating RNA bound in the cavity. Ends of RNA are labeled. c, end view of complex, with surface view of YicC colored by chain. d, arginine resides help bind the RNA backbone. The arginine sidechains are colored according to chain. Selected arginines are shown that are in proximity of the phosphate backbone. e, catalytic site of YicC. The proposed cleavage bases, C3 and A4 are colored in yellow. Water molecules in structure are shown as red spheres, W1 and W2. Essential glutamates are shown, colored according to chain.

Figure 3. Fluorescent assay of RNA cleavage.

a, Time course of YicC activity. RNA concentration was 600 nM; YicC protein concentration was as indicated above each set of reactions. Reactions were incubated at 37°C and aliquots were removed at times indicated above each lane. Control lane (−), no YicC protein added. Migration of full-length RNA oligo (A) and cleavage products (C and B) indicated at left. b, Sequence and secondary structure of 26-mer RNA used in the cryo-EM study. The same 26-mer, but with a 5’-IR800 fluorophore, was used for cleavage assays. Two sites of YicC cleavage are shown. c, Predicted cleavage fragments of 26-mer RNA. Fragments C, D, and E were confirmed by mass spectroscopy. d, YicC cleavage of 26-mer and 36-mer. RNA concentration in each case was 600 nM; YicC concentration was 20 nM for the 26-mer reaction and 100 nM for the 36-mer reaction.

Figure 4. Screening of YicC residues required for cleavage.

26-mer RNA concentration was 600 nM; YicC protein concentration was 20 nM. Reactions were incubated at 37°C and aliquots were removed at 1 min and 16 min. The YicC residue that was mutated to alanine is indicated for each mutant protein. Control lane (−), no YicC protein added. Migration of full-length RNA oligo (A) and cleavage products (C and B) indicated at left.

Figure 5. Analysis of YicC mutant binding to RNA by fluorescence anisotropy.

Binding constants were measured using a fixed concentration of 5’-Cy3 labeled RNA 36-mer and varied concentrations of wild-type or mutant YicC protein. Fluorescent polarization was measured on a multimode plate reader. The binding constants are shown in Table S2. n=3 for each data point, shown with error bars for standard deviation.