Abstract:
Through the past decades, the model of a cotranslational import of nuclear encoded proteins into cellular organelles moved from the edge to the spotlight of attention. Indispensable for such a process is the correct targeting of an according mRNA or the ribosome-mRNA complex. Involved proteins and cis-elements are yet widely unknown.
This work concentrates on the mitochondrial localized SOD2 mRNA. It aims to identify its interactome and especially the interacting factors at the mitochondrial surface by applying the RNA-BioID method on a bait mRNA based on the human superoxide dismutase 2 mRNA.
The expression of all components – the SOD2MS2 mRNA and a 2xMCP-eGFP-BirA* fusion protein – could be successfully validated in transiently transfected HeLa S3 cells. In addition, the nuclear localization of the fusion protein and its distribution into cytoplasmic granules upon transfection with a SOD2MS2 encoding vector (SOD2MS2) were visualized.
The fusion protein 2xMCP-eGFP-BirA* and a variant, 1xMCP-eGFP-BirA*, were integrated into the genomes of HeLa EM2-11ht and U2OS T-Rex cells and expressed under a promoter regulated by tetracycline response elements. Validation of the system was not fully performed for both cell lines, but the doxycycline-dependent expression of the fusion proteins could be demonstrated in both cell lines. The correct localization of the construct was verified via fluorescence microscopy.
Finally, RNA-BioID was performed in U2OS1xMCP-eGFP-BirA* cells. SOD2MS2 transfected cells as well as untransfected controls were induced with doxycycline and maintained in biotin containing medium. Biotinylated proteins were determined by mass spectrometry. Although not a quantitative method, the mass spectrometry data allowed the identification of several candidates for proteins interacting with SOD2MS2 mRNA. Among them were cytosolic proteins involved in mRNA regulation and transport as CLUH, FXR1, PABP, STAU1, FUBP3 and LARP1. In addition, proteins involved in membrane and vesicle trafficking passed our threshold, as well as the TOM-complex associated cytosolic protein TOM34. Potential interactors at the mitochondrial surface were ATAD3, Rab5c and IGF2BP2. Other proteins of interest might be LRPPRC and the so far uncharacterized LRRC40. All these candidates must be carefully evaluated in further experiments to elucidate the interacting factors of SOD2 mRNA and the mode of SOD2 protein import into mitochondria, in particular since SOD2 mRNA was lately not found associated to the mitochondrial surface and no component of TOM complex, the main mitochondrial protein importer, is found in our dataset.
Further experiments will have to address an improvement of the RNA-BioID system in stably expressing cells and profound validation as well as the characterization of the possible candidates. If performed carefully, they may build the foundations to develop drugs for diseases related misregulation of SOD2.
mRNA transport