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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 Nuclear gene inactivation: general framework Nuclear gene inactivation: RNA processing U2 snRNA Nuclear gene inactivation: RNA processing Rnt1p dsRNAse Biochemistry (2008) 47: 8514 Inducible gene inactivation: RNA folding Inducible gene inactivation: using guide RNAs “Short RNA guides cleavage by eukaryotic RNase III.” PLoS ONE. (2007) 2(5):e472 Inducible inactivation system for mitochondrial gene expression mt gene +dox mt gene Inducible inactivation system for mitochondrial gene expression Part1: mtRNT1 mt gene FY2068 MAT ura3-52 his3200 leu2 1 lys2-128 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 Is mtRNT1 induced + dox? anti-Rnt1 Western 1 2 3 4 5 “- mtRNT” 75 50 kDa “+ mtRNT” 1 Marker 2 - mtRNT, - dox 3 + mtRNT, - dox 4 - mtRNT, + dox 5 +mtRNT, + dox 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 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 MH36 (YPD) DAPI Alexa 100X total magnification 1:4000 1° Ab Is mtRNT1 localized to mt? “+ mtRNT” - dox DAPI Alexa + dox +mtRNT1 -dox DAPI DIC 100X total magnification 1:4000 1° Ab Alexa +mtRNT1 -dox DAPI Alexa 100X total magnification 1:4000 1° Ab +mtRNT1 +dox DAPI DIC 100X total magnification 1:4000 1° Ab Alexa +mtRNT1 +dox DAPI Next: Try GFP fusion to mtRNT1? Alexa 100X total magnification 1:4000 1° Ab Are there consequences of induction/localization? = - mtRNT +dox = + mtRNT +dox Agilent 4x44K spotted array, 3DNA labeling mtRNT: most affected genes, pt 1 higher no mtRNT I-SceII, encoded by intron within the mitochondrial COX1 gene mfold.bioinfo.rpi.edu/ mtRNT: most affected genes, pt 1 higher no mtRNT Dubious ORF in mt genome within RPM1 gene mfold.bioinfo.rpi.edu/ mtRNT: most affected genes, pt 1 higher no mtRNT I-SceIV, encoded by intron within the mitochondrial COX1 gene Sequence is 9350 bases…too long for mfold (limit 800) mtRNT: most affected genes, pt 1 higher no mtRNT F0-ATP synthase subunit 9 encoded on the mitochondrial genome mfold.bioinfo.rpi.edu/ Ask: OligomycinR? mtRNT: most affected genes, pt 2 higher + mtRNT RNT1 upregulated mtRNT: most affected genes, pt 2 higher + mtRNT RNA-binding protein End of first part