We developed a new ribosome profiling approach that leverages the principles of microfluidics and isotachophoresis: Ribo-ITP (Ozadam et al., Nature):
Previous studies had been restricted to bulk measurements precluding precise determination of translation regulation including allele-specific analyses. To address this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allelic-biased expression. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.
We use Ribo-ITP to investigate diverse biological questions, often in close collaboration with domain experts. Recent highlights include:
- Characterization of allelic variants in the ribosome biogenesis factor AIRIM in human cerebral organoids (Ni et al., Nature Cell Biology, in press).
- Application of Ribo-ITP in C. elegans to map dynamic changes in the translation of maternal mRNAs during the first four cell cycles of embryogenesis (Shukla et al., Cell Reports, in press).
- Use of selective ribosome profiling in yeast to show that that Reh1-bound ribosomes accumulate near start codons. Our data suggest that Reh1 is displaced by the elongating nascent peptide and is the final assembly factor released from the 60S ribosomal subunit during its first round of translation (Musalgaonkar et al., Nature Communications).