Cleavage region organizes the structural architecture of the B2 SINE ribozyme

The SINE-encoded B2 retrotransposon is an RNA Polymerase III transcript that gets upregulated during various cellular stress responses. The B2 noncoding RNA can directly inhibit RNA Polymerase II, leading to a significant downregulation of transcripts during stress. Our recent findings have shown that B2 is a self-cleaving epigenetic ribozyme and that cleavage can be induced by interactions with epigenetic factors, co-regulating its function across distinct chromatin-binding target loci. Here, by integrating RNA chemical probing, small angle X-ray scattering, and 3D motif modeling, we determine structural ensemble-to-function relations for the B2 SINE ribozyme RNA. Perturbations of the RNA suggest that the B2 SINE ribozyme has a well-defined secondary structure and dynamic tertiary structure that both critically depend on the presence of the active site of cleavage. In an RNA engineering approach, we examine the effect of point mutations, deletions of the main cleavage site and deletions of the cleavage domain on the structural ensemble of the RNA. By combining this with functional data, we obtain relations of structural ensembles to various functional states. This perturbative approach serves as a template to unravel the relation of structural ensembles to functional states for other ncRNA and mRNA systems.