An ultra-low-input RNase footprinting assay to quantify cytosolic and mitochondrial translation simultaneously

Ribosome profiling isolates ribosome-protected fragments for sequencing and reveals active translation at the single-nucleotide resolution in vivo. It represents a valuable approach to study various aspects of protein synthesis, such as the regulation of translation efficiency, alternative translation initiation, ribosome elongation and pausing, codon usage, and identifying non-canonical open reading frames and micropeptides (<100 amino acids) encoded in a genome. Thus, it provides unique molecule insights of translational control, which could not be achieved by other genomic technologies, such as mass spectrometry or polysome profiling. However, current ribosome profiling protocols generally use complicated experimental procedures to isolate ribosome-RNA complexes such as though a sucrose cushion, and require millions of input cells. This technical barrier has prevented its application to examing translation profiles of primary physiological tissue samples with low-input cell numbers. To tackle this long-standing challenge, here we propose to develop an ultra-low-input RNase footprinting approach for the rapid quantification of cytosolic and mitochondrial translation simultaneously. Our method simplified the experimental procedure to select ribosome footprints based on optimized RNase digestion. My lab has made the assay work well for as few as 1,000 cultured cells. In this proposal, we aim to further develop the assay and make it work robustly for a small amount of primary tissue samples (Aim 1). Furthermore, we will apply the method to map the RNA translation landscape of rare progenitor cells and differentiated cell types during hematopoiesis (Aim 2). The results will reveal novel mechanisms mediating the translational control underlying hematopoietic cell fate decisions. Finally, by leveraging that our assay provides a simplified method to study mitochondrial translation, we will use it to examine the heterogeneity of mitochondrial translation machinery and build the functional network mediated by individual factors (Aim 3). Altogether, the refined ultra-low-input RNase footprinting method developed in this proposal will be a valuable tool and can be widely used to study the translational regulation underlying complex physiological conditions from normal development to disease progression.