Peroxidase- and photosensitizer-mediated proximity labeling for mitochondria-associated transcriptome analysis in live cells

Abstract

The precisely subcellular and sub-organellar RNA localization at a right time is fundamental for their biological functions in eukaryotic cells. Among various organelles, semi-autonomous mitochondria have received growing attention in recent years. To profile mitochondria-associated transcriptomes with high spatial resolution in live cells, proximity-based labeling approaches have rapidly evolved over the past decade. In this thesis, chapter 1 presented an overview of the current RNA proximity labeling strategies, mitochondria-associated transcriptomes and their underlying roles, and the representative applications of mitochondria targeting small molecules. Though APEX2 mediated proximity labeling has been extensively implemented for RNA subcellular localization in live cells, the relatively low labeling efficiency of the original biotin phenoxyl radical towards RNAs still limits its compatibility with other characterization methods. Thus, in chapter 2, a set of clickable APEX2 probes were explored to improve the RNA labeling efficiency by balancing reactivity, lipophilicity, and hydrophilicity. Among them, we verified the great sensitivity and specificity of APEX2 labeling with Ph_N3 at the transcriptome level in live HEK 293T cells stably expressing APEX2 enzyme in the mitochondrial matrix. Moreover, Ph_N3 provided two biotinylation routes for downstream analysis and worked orthogonally with singlet oxygen (1O2) based method to enable the dual location labeling at the imaging level in living cells. Chapter 3 focused on the development of RNA proximity labeling approach based on aggregation induced emission (AIE) type photosensitizers with great mitochondria targeting ability. We compared the in vitro optical properties and photosensitization processes as well as the in cellulo mitochondrial specificity and RNA labeling performance of our four AIE photosensitizers with classic donor-acceptor backbones. The high-throughput sequencing analysis with TFPy-mediated RNA labeling in live HeLa cells showed that both most mitochondria encoded RNAs typically inside mitochondria and some nuclear derived RNAs at the outer mitochondrial membrane (OMM) and the interacting organelles were included in our datasets. This small molecule-based proximity labeling method bypasses complex genetic manipulation and transfection steps, being readily incorporated for diverse research aims. In chapter 4, we turned our attention to OMM, which not only acts as the first barrier for entry of nuclear encoded transcripts and proteins but also plays crucial roles in interacting with other organelles. HaloTag protein ligand linked dibromofluorescein (Halo-DBF) was utilized to decipher the transcriptomes at the OMM under basal and two classical cytoskeletal disruption conditions in live U2OS cells stably expressing TOMM20-HaloTag fusion protein. With the great targeting performance of Halo-DBF towards OMM, a list of OMM libraries were constructed and the differential expression analysis of our three datasets reflected the evident overlap with the published OMM data as well as the distinct transcriptome alterations upon cytoskeletal perturbations. Due to global cellular responses towards the cytoskeletal disruptions, the current work may not fully reveal the impacts of cytoskeletal perturbation on the OMM localized transcriptomes, and further experimental evidence from other aspects would be required to better comprehend the RNA localization patterns at the OMM.