To examine the putative translation of circRNAs, we set up the Ribosomal Footprinting (RFP) methodology using fly heads. As expected, we obtained strong coverage of the known open reading frames and their 5’ UTRs (4A), providing the first RFP dataset in adult fly tissue. As expected, we observed phasing of the reads (Figure 4A), which indicates that our data can be exploited to assert translation in a candidate region. Using this approach and a newly developed algorithm to score start, stop and ORF sequences, we were able to identify hundreds of new ORF expressed in Drosophila heads, most of which are upstream ORFs. Interestingly, we found RFP reads originating from circRNAs. In most cases the number of RFP reads encompassing circRNA junctions are in the same scale as the RFP exon-junction reads of the corresponding linear mRNA. The fly head RFP identified 122 ribosome-associated circRNAs (ribo-circRNA). Only patterns in RFP reads encompassing the backsplicing junction can be unequivocally attributed to circRNA translation, which greatly diminishes our capacity to look for start codons, stop codons and significantly diminishes the power for detecting ribosome phasing encompassing the ORF. Despite these limitations, we observed a high stop codon score for the most abundant ribo-circRNA, circMbl. This result supports translation of circMbl in fly heads. To obtain additional data on translation of circRNAS, we utilized a fly strain carrying a transgene for expressing EGFP-tagged ribosomes. We immunopurified the tagged ribosomes and analyzed the RNA bound to them. We measured the relative IP enrichment. Indeed, we found a strong enrichment for four ribo-circRNAs assayed, demonstrating their association with ribosomes. To distinguish between associations of ribosomes with linear and circular RNA molecules we treated ribosome-bound RNAs with RNAseR. Indeed, we found that the four assayed ribo-circRNAs are strongly resistant to RNAseR, demonstrating that they are bound to the ribosome as circular RNA molecules. Importantly, we observed putative proteins produced by different circRNAs generated from the mbl locus by Western blot and by mass spectrometry
Pamudurti N., Bartok O., Jens O., Ashwal-Fluss R., Stottmeister C., Ruhe L., Hanan M., Wyler E., Perez-Hernandez D., Ramberger E., Shenzis S., Samson M., Dittmar G., Landthaler M., Chekulaeva M., Rajewsky N. and Kadener S. “Translation of circRNAs”. Molecular Cell 66(1), 9-21 (2017). [Web]
