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Synthetic Systems for Teaching and Learning Winston Retreat June 25th, 2007 Natalie Kuldell.

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Presentation on theme: "Synthetic Systems for Teaching and Learning Winston Retreat June 25th, 2007 Natalie Kuldell."— Presentation transcript:

1 Synthetic Systems for Teaching and Learning Winston Retreat June 25th, 2007 Natalie Kuldell

2 Regulating RNA degradation in yeast mitochondria Undergraduate teaching with SAGA deletions Unreal Irrational

3 Why hack the yeast mitochondria? “ (we often) imagine the mitochondrion as a lonely participant in the cell, working tirelessly to produce the energy required for life.” McBride et. al. Curr Biol Other mt functions coordinates with nuclear gene expression (disease/aging) spatially isolated enzymatic reaction center viability on nonfermentable carbon sources

4 ? = Hacking yeast mitochondria Wish list (incomplete) 1. Orthogonal draw from different pools of reagents 2. Decoupled run system independent of growth rate 3. Generic run same system in different chassis 4. Tunablevary operation at will

5 Current contents: in mt from mt mt genome includes 8 protein coding genes 7 oxphos, 1 riboprot 2 rRNAs 24 tRNAs mt promoters TATAAGTA (+1) mt RNAP RPO41 = catalytic subunit MTF1 = specificity factor nuclear- encoded

6 Targeted mtRNA degradation Part 3: dsRNase Part 1: mRNA target e.g. mtGFP Part 2: guide RNA

7 Catala et al, MCB (2004) 15:3015 Snapshot of wild type role for Rnt1 Localized to the nucleus even when overexpressed

8 Snapshot of wild type role for Rnt1 Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs) Localized to the nucleus even when overexpressed Henras et al.RNA (2004) 10: 1572

9 Snapshot of wild type role for Rnt1 Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs) Localized to the nucleus even when overexpressed Processes some coding RNA, e.g. Mig2 Ge et al, Current Biology (2005) 15:140

10 Catala et al, MCB (2004) 15:3015 Snapshot of wild type role for Rnt1 Processes some noncoding RNAs (U2 snRNA, U3 snoRNPs) Localized to the nucleus even when overexpressed Processes some coding RNA, e.g. Mig2 Needed for normal cell cycle progression

11 tTA CMV 2x tetO CYC1 pRS41n modified RNT1 Expression vector for mitochondrial Rnt1

12 pRS41n RNT1 signal sequence+ epitope tag ∆NLS (11 aa) ∆NLS in Henras et al RNA (2004) 10:1572

13 Initial experiments with mtRnt1 1. Expression? by Western with epitope Ab 2. Phenotypes? Respiration, growth, existing markers 3. Overall? Microarray wt vs mtRnt

14 Targeted mtRNA degradation Part 3: dsRNase Part 1: mRNA target e.g. mtGFP Part 2: guide RNA

15 Protein import into mitochondria Pfanner and Geissler Nat Rev (2001) 2:339

16 RNA import into mitochondria “poorly understood”/”mechanisms appear to differ”

17 RNA import into mitochondria “poorly understood”/”mechanisms appear to differ” ~all mt tRNAs encoded on nuclear genome RNA receptor (“RIC”) in mt membrane Entelis et al Gene Engineering: Principles and Methods (2001) 24:191

18 RNA import into mitochondria “poorly understood”/”mechanisms appear to differ” no mt tRNAs encoded by mt RIC + ytRNA--> repair mt defect in human cell line Mahata et al Science (2006) 314:471

19 RNA import into mitochondria “poorly understood”/”mechanisms appear to differ” all but one tRNA encoded on mt genome import depends on protein import

20 Specialized import into mitochondria protein:RNA conjugate Piggyback on tRNA import Bind to mtRNA binding protein

21 Regulating RNA degradation in yeast mitochondria Undergraduate teaching with SAGA deletions Unreal Irrational

22 Expression Engineering Experiment Day 1Day 2Day 3 Day 4Day 5Day 6 RT

23 Subunit Deleted? ADA31/1 GCN51/1 SPT3 (3 groups)3/3 SPT8 (3 groups)0/3 UBP8 (2 groups)2/2 SUS1 (2 groups)2/2 FY2068 A ura3-52 his3∆200 leu2∆1 lys2-128 

24 Subunit Deleted? ADA31/1 GCN51/1 SPT3 (3 groups)3/3 SPT8 (3 groups)0/3 UBP8 (2 groups)2/2 SUS1 (2 groups)2/2 FY2068 A ura3-52 his3∆200 leu2∆1 lys2-128  NY389  ura3-52 his4-917  leu3∆1 trp1-63 spt8∆320::LEU2

25 Day 3

26 wt/sus1∆ Andrew Ji and Kate Broadbent, W/F Team Blue, Spring ‘07 wt/sgf73∆ teacher Follow-up with microarray

27 Follow-up with spot tests

28 Hi Natalie, I've attached my rewrite. Thanks! See you tomorrow, Andrew P.S. This was one of the most time-consuming assignments I've ever had to do, yet it was easily the most fun and rewarding thing I've ever accomplished for any school-related project. Follow-up with spot tests

29 Andrew Ji and Kate Broadbent May 10, 2007 Sus1’s role in SAGA-dependent gene motility, transcription, and expression under different cellular conditions

30 From: Neal Lerner Subject: Re: 109 writing assignment Date: Thu, 11 Jan :07: To: natalie kuldell Natalie, as I prepare to give a writing-across-the-curriculum talk next week, I came across this quote from John Bean: WAC is about creating opportunities for students to have an "authentic desire to converse with interested readers about real ideas." Now, in most school settings that's pretty darn hard to achieve, but I think when students have the chance to write/talk about lab work and ideas they find interesting (as in ), we have a shot at it. See you on the 22nd. Neal

31 the end

32 Current contents: in mt from nucleus nuclear genome sends ~750 proteins to mt 87 of these are putative proteins of no known function


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