Discovery of Stimulus-Responsive Microproteins in Human Microglia

PROJECT SUMMARY This R21 proposal outlines a collaborative research initiative aimed at elucidating how human microglia-like cells (iMGLs) leverage mRNA translational control mechanisms to respond to environmental cues. Human microglia must leverage significant protein diversity to mount immune responses and shape behavior in response to external stimuli, but the role of mRNA translation in microglial function is poorly understood. Further, mRNA translation is far more complex than previously appreciated, as translation occurs within regions of the genome annotated as non-coding – so-called non-canonical open reading frames (ncORFs). ncORFs include small open reading frames (sORFs) less than 300 nucleotides that encode microproteins less than 100 amino acids. Microproteins have been implicated as critical biological effectors of the immune response, but the role of such microproteins in human microglia is unknown. We hypothesize that microproteins may be an important and indispensable component of human microglia function and the response to environmental stimuli. As such, we will define the translational landscape – the “translatome” – of iMGLs using ribosome profiling, which allows us to map translation at single nucleotide resolution. We will define open reading frames in iMGLs, and identify both novel sORFs and putative microproteins. We will similarly use ribosome profiling to identify translational responses to external stimuli, such as myelin debris and amyloid. We will confirm our findings using size-selected proteomics to validate stimuli-dependent human microglial microproteins. The knowledge derived from this research plan will not only enhance our understanding of the mechanisms through which human microglia respond to the local microenvironment, but will also identify candidate microproteins that can be functionally interrogated as critical effectors of human microglia function.