Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation

We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repression of cytosolic translation after TUFM perturbation. We show the assay is applicable to small amounts of primary tissue samples with low protein synthesis rates, including snap-frozen tissues and immune cells from an individual's blood draw. We showed its feasibility to characterize the personalized immuno-translatome. Our analyses revealed that thousands of genes show lower translation efficiency in monocytes compared with lymphocytes, and identified thousands of translated noncanonical open reading frames (ORFs). Altogether, our RNase footprinting approach opens an avenue to assay transcriptome-wide translation using low-input samples from a wide range of physiological conditions.

Automated summary

We developed a low-input RNase footprinting technique for rapid quantification of ribosome-protected fragments, applicable to small samples and translation studies under different physiological conditions.