Comprehensive Identification of Translatable Circular RNAs Using Polysome Profiling

Bio Protoc. 2021 Sep 20;11(18):e4167. doi: 10.21769/BioProtoc.4167.


Circular RNAs (circRNAs), a special type of RNAs without 5'- and 3'-ends, are widely present in eukaryotes and known to function as noncoding RNAs to regulate gene expression, including as miRNA sponges. Recent studies showed that many exonic circRNAs, generated by back-splicing of pre-mRNAs, can be translated in a cap-independent fashion through IRESs or m6A RNA methylation. However, the scope of the translatable circRNAs and the biological function of their translation products are still unclear in different cells and tissues. Ribosome footprinting and proteomic analysis were usually used to globally identify translatable circRNAs. However, both methods have low sensitivity due to the low efficiency in the discovery of circRNA specific reads or peptides (i.e., the back-splicing junctions are difficult to recover by the short reads of ribosome footprinting and the limitation of proteomic analysis). Here, we described an alternative method to identify translatable circRNAs using polysome profiling and circRNA-seq. Generally, polysome-associated RNAs were separated with sucrose gradients. Then polysome-bound circRNAs were enriched by an RNase R treatment and identified through paired-end deep sequencing. Thus, this method is more sensitive than ribosome footprint and proteomic analyses for the identification of translatable circRNAs.

Keywords: Polysome profiling; RNA-seq; RNase R; back-splicing junction; circRNA translation.