UCLA

PNPase is a conserved 3’-5’ exoribonuclease localized in the mammalian mitochondria. Its expression is essential for embryonic development and mitochondrial homeostasis. PNPase has been recently implicated in regulating mitochondrial RNA import. Utilizing yeast mutants and knockdown systems in human cell lines, we show that porin or VDAC may function as a possible import channel in the mitochondria. The radiolabeled RNA that is imported into the purified mammalian mitochondria crosslinks specifically with VDAC1 suggesting that the imported RNA may go through this outer membrane channel. In order to further study the role of PNPase, we attempted to create a knockout cell line by adjusting the culture conditions to allow cells to survive without functional mitochondria. Interestingly, the resulting cell lines did not have any mitochondrial DNA shown by the lack of signal from the PCR performed using probes designed to detect the mitochondrial DNA, and these cells also did not respire. Overall, PNPase knockout cells showed a transcriptional profile that was similar to rho0 cells that lack mitochondrial DNA. Further, PNPase knockout cell lines showed changes in genes involved in cholesterol biosynthesis and regulating neuronal function. This study underscores the unexpected role of PNPase in affecting mitochondrial DNA maintenance. Finally, we characterized endogenous RNA targets of PNPase, its effect on steady state levels of mitochondrial RNA when PNPase expression is knocked down, and its interaction partners. We find that PNPase binds all mitochondrial tRNAs, mRNAs and rRNAs but specific regions that include tRNAs are enriched in the PNPase CRAC assay. When PNPase expression is knocked down, the steady state levels of sense strand RNAs do not change significantly, but specific antisense strand RNAs transcribed from the mitochondrial DNA accumulate at high levels. Using the proximity based BIOID2 biotin labeling system and mass spectrometry analysis, we identified that PNPase may interact with mitochondrial ribosome assembly factors such as FASTKD2, DDX28, and DHX30, and other members of the FASTKD family. Our research suggests that PNPase plays an essential role in the mitochondria where it may regulate mitochondrial DNA maintenance and mitochondrial RNA levels, and it interacts with specific mitochondrial ribosome assembly factors and RNA processing proteins.