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Evaluating Inducible Inactivation Systems for Mitochondrial Gene Regulation in Summer Research

This summer, I focused on evaluating an inducible inactivation system designed to regulate mitochondrial gene expression. My research aimed to explore the unintended consequences of TAP-tagging and investigate nuclear gene inactivation mechanisms. I conducted a detailed examination of RNA processing techniques involving U2 snRNA and Rnt1p, while also assessing the localization and induction of mtRNT1 in mitochondria. This work builds upon previous frameworks in RNA processing and aims to advance our understanding of mitochondrial gene regulation.

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Evaluating Inducible Inactivation Systems for Mitochondrial Gene Regulation in Summer Research

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  1. What I wanted to do this summer Evaluate inducible inactivation system designed to regulate mitochondrial gene expression Examine unintended consequences of TAP-tagging What I really did this summer

  2. Nuclear gene inactivation: general framework

  3. Nuclear gene inactivation: RNA processing U2 snRNA

  4. Rnt1p dsRNAse Nuclear gene inactivation: RNA processing Biochemistry (2008) 47: 8514

  5. Inducible gene inactivation: RNA folding

  6. Inducible gene inactivation: using guide RNAs “Short RNA guides cleavage by eukaryotic RNase III.” PLoS ONE. (2007) 2(5):e472

  7. Inducible inactivation system for mitochondrial gene expression +dox mt gene  mt gene

  8. Inducible inactivation system for mitochondrial gene expression Part1: mtRNT1 mt gene FY2068 MAT ura3-52 his3200 leu2 1 lys2-128 

  9. Inducible inactivation system for mitochondrial gene expression Part1: mtRNT1 • Is mtRNT1 induced +dox? • Is mtRNT1 localized to mt? • Are there consequences of induction/localization? mt gene

  10. Is mtRNT1 induced + dox? anti-Rnt1 Western 1 2 3 4 5 “- mtRNT” 75 50 kDa 1 Marker 2 - mtRNT, - dox 3 + mtRNT, - dox 4 - mtRNT, + dox 5 +mtRNT, + dox “+ mtRNT”

  11. Is mtRNT1 localized to mt? “The results presented so far indicate that Rnt1p is a nuclear enzyme distributed throughout the nucleus that also strongly accumulates within a nucleolar structure corresponding to the site of transcription of the rDNAs.” “A cotranscriptional model for 3'-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor” RNA (2004) 10(10):1572

  12. Is mtRNT1 localized to mt? “A cotranscriptional model for 3'-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor” RNA (2004) 10(10):1572

  13. MH36 (YPD) DAPI Alexa 100X total magnification 1:4000 1° Ab

  14. Is mtRNT1 localized to mt? “+ mtRNT” - dox + dox DAPI Alexa

  15. +mtRNT1 -dox DAPI DIC Alexa 100X total magnification 1:4000 1° Ab

  16. +mtRNT1 -dox DAPI Alexa 100X total magnification 1:4000 1° Ab

  17. +mtRNT1 +dox DAPI DIC Alexa 100X total magnification 1:4000 1° Ab

  18. +mtRNT1 +dox DAPI Next: Try GFP fusion to mtRNT1? Alexa 100X total magnification 1:4000 1° Ab

  19. Are there consequences of induction/localization? = - mtRNT +dox = + mtRNT +dox Agilent 4x44K spotted array, 3DNA labeling

  20. I-SceII, encoded by intron within the mitochondrial COX1 gene mfold.bioinfo.rpi.edu/ mtRNT: most affected genes, pt 1 higher no mtRNT

  21. Dubious ORF in mt genome within RPM1 gene mfold.bioinfo.rpi.edu/ mtRNT: most affected genes, pt 1 higher no mtRNT

  22. I-SceIV, encoded by intron within the mitochondrial COX1 gene mtRNT: most affected genes, pt 1 higher no mtRNT Sequence is 9350 bases…too long for mfold (limit 800)

  23. F0-ATP synthase subunit 9 encoded on the mitochondrial genome mfold.bioinfo.rpi.edu/ mtRNT: most affected genes, pt 1 higher no mtRNT Ask: OligomycinR?

  24. RNT1 upregulated mtRNT: most affected genes, pt 2 higher + mtRNT

  25. mtRNT: most affected genes, pt 2 higher + mtRNT RNA-binding protein

  26. End of first part

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