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

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

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

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

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

11.  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 “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.”

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

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

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

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

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

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

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

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

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

22. 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)

23. 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?

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

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

26. End of first part