RNase P cleavage of pseudoknot substrates reveals differences in active site architecture that depend on residue N-1 in the 5' leader.

We show that a small biotin-binding RNA aptamer that folds into a pseudoknot structure acts as a substrate for bacterial RNase P RNA (RPR) with and without the RNase P C5 protein. Cleavage in the single-stranded region in loop 1 was shown to depend on the presence of a RCCA-motif at the 3' end of the substrate. The nucleobase and the 2'hydroxyl at the position immediately 5' of the cleavage site contribute to both cleavage efficiency and site selection, where C at this position induces significant cleavage at an alternative site, one base upstream of the main cleavage site.

Efficient 3'-pairing renders microRNA targeting less sensitive to mRNA seed accessibility.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3'-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3'-pairing and secondary structures in mRNAs on repression efficiency.