Ornate, large, extremophilic (OLE) RNA forms a kink turn necessary for OapC protein recognition and RNA function

Abstract

Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA. Disruption of the genes for either OLE RNA, OapA, or OapB result in the inability of cells to overcome cold, alcohol, or Mg2+ stresses. In the current study, we used invivo crosslinking followed by OLE RNA isolation to identify the protein YbxF as a potential additional partner in the OLE RNP complex. Notably, a mutation in the gene for this same protein was also reported to be present in a strain wherein the complex is nonfunctional. The B.halodurans YbxF (herein renamed OapC) is homologous to a bacterial protein earlier demonstrated to bind kink turn (k-turn) RNA structural motifs. Invitro RNA-protein binding assays reveal that OLE RNA forms a previously unrecognized k-turn that serves as the natural binding site for YbxF/OapC. Moreover, B.halodurans cells carrying OLE RNAs with disruptive mutations in the k-turn exhibit phenotypes identical to cells lacking functional OLE RNP complexes. These findings reveal that the YbxF/OapC protein of B.halodurans is important for the formation of a functional OLE RNP complex.