Characterization of Circular RNAs (circRNA) Associated with the Translation Machinery

A substantial proportion of the currently annotated genes in eukaryotes are proposed to function as RNA molecules (>200 bp) with no significant protein coding potential, currently classified as long noncoding RNAs (lncRNA). A distinct subgroup of lncRNAs is circular RNAs (circRNAs), which can be easily identified by unique junction reads, resulting from their biogenesis. CircRNAs are largely cytosolic and thought to either code for micro-peptides or facilitate gene regulation by sequestering microRNAs (miRNAs) or RNA-binding proteins (RBPs) from their targets. Interrogation of the interaction of circRNAs with cellular macromolecular machineries could indicate their mode of action. Here, we detail a sucrose gradient-based method to pinpoint association of a given circRNA (or any transcript of interest) with distinct ribosomal fractions. This method can evaluate the coding potential of candidate circRNAs (or any transcript of interest) and its association with the translation machinery.