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Biochemical Characterization of the Endoribonuclease, EndoU, and its Role in the Negative Selection of Developing Thymocytes

  • Author(s): Dias, Kristen
  • Advisor(s): Karginov, Fedor V
  • et al.
Abstract

RNA binding proteins (RBPs) are an important but a poorly characterized group of proteins that affect all stages of the lifecycle of RNA transcripts, also known as post-transcriptional regulation. Within this class of proteins are RNA endonucleases. These RNA endonucleases have been shown to, both, upregulate and downregulate the propensity of a cell to undergo programmed cell death, known as apoptosis. The endoribonuclease poly-U specific, EndoU, is one such RNA endonuclease that has been implicated in promoting apoptosis but has not been fully characterized to show how it elicits this cell death. EndoU shows a tissue expression pattern in highly transient cells that are primed for cell death. Here, EndoU was investigated using the immature thymocyte cell line, VL3-3M2, at the biochemical level to show that the mammalian EndoU used calcium, likely as an allosteric cofactor, for its activity. Further biochemical characterization revealed that it was regulated by a large-transiently interacting cofactor that broadened its activity. This cell line was also used to characterize the cellular role for EndoU and was found that it positively regulated pro-apoptotic, calcium homeostasis, and cell motility related genes. Further support was given to this regulation by EndoU, when protein sequencing of the likely EndoU complex was shown that it included the pro-apoptotic proteases, calpain 1 and 2. The complex also likely included RBPs that bound ribosomal RNA and were involved in tRNA processing, an indication that EndoU may cleave these RNAs during a calcium burst. Finally, it was shown that, in mice, EndoU promoted the negative selection of self-reactive thymocytes but not death by neglect, a result not previously seen before. While EndoU could be characterized further, the results showed that this enzyme was a highly regulated endoribonuclease that, in vitro, did not prefer a specific substrate, but, in vivo, it was shown to enhance apoptosis likely due to the release of these regulating factors.

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