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).
- 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).
- Application of Ribo-ITP to uncover non-canonical translation events in ultra-low-input samples (Ghatpande et al., bioRxiv). Using Ribo-ITP, we profiled ribosome occupancy in microdissected hippocampal tissues and single mouse embryos, revealing thousands of translated short open reading frames (“translons”). Functional assays confirmed translation of selected translons, extending the Ribo-ITP platform to define hidden layers of the translatome in rare cell populations.