What I wanted to do this summer

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