Synthetic Systems for Teaching and Learning

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Synthetic Systems for Teaching and Learning

• Winston Retreat
• June 25th, 2007

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Unreal Irrational
Undergraduate teaching with SAGA deletions
Regulating RNA degradation in yeast mitochondria
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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 2006

• Other mt functions
• coordinates with nuclear gene expression
  (disease/aging)
• spatially isolated enzymatic reaction center
• viability on nonfermentable carbon sources

http//grocs.dmc.dc.umich.edu/gallery/organelle/In
terface2
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Hacking yeast mitochondria
?

• Wish list (incomplete)
• Orthogonal draw from different pools of reagents
• Decoupled run system independent of growth rate
• Generic run same system in different chassis
• Tunable vary operation at will

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Current contents in mt from mt

• mt genome includes
• 8 protein coding genes
• 7 oxphos, 1 riboprot
• 2 rRNAs
• 24 tRNAs

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Targeted mtRNA degradation
Part 3 dsRNase
Part 2 guide RNA
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Snapshot of wild type role for Rnt1

• Localized to the nucleus even when overexpressed

Catala et al, MCB (2004) 153015
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Snapshot of wild type role for Rnt1

• Localized to the nucleus even when overexpressed

• Processes some noncoding RNAs (U2 snRNA, U3
  snoRNPs)

Henras et al.RNA (2004) 10 1572
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Snapshot of wild type role for Rnt1

• Localized to the nucleus even when overexpressed

• Processes some noncoding RNAs (U2 snRNA, U3
  snoRNPs)

• Processes some coding RNA, e.g. Mig2

Ge et al, Current Biology (2005) 15140
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Snapshot of wild type role for Rnt1

• Localized to the nucleus even when overexpressed

• Processes some noncoding RNAs (U2 snRNA, U3
  snoRNPs)

• Processes some coding RNA, e.g. Mig2

• Needed for normal cell cycle progression

Catala et al, MCB (2004) 153015
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Expression vector for mitochondrial Rnt1
CMV
CYC1
tTA
modified RNT1
2x tetO
pRS41n
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Expression vector for mitochondrial Rnt1
signal sequence
epitope tag
?NLS (11 aa)
RNT1
pRS41n
?NLS in Henras et al RNA (2004) 101572
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Initial experiments with mtRnt1

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

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Targeted mtRNA degradation
Part 3 dsRNase
Part 2 guide RNA
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Protein import into mitochondria
Pfanner and Geissler Nat Rev (2001) 2339
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RNA import into mitochondria
poorly understood/mechanisms appear to differ
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RNA import into mitochondria
poorly understood/mechanisms appear to differ
all mt tRNAs encoded on mt genome
RNA receptor (RIC) in mt membrane
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RNA import into mitochondria
poorly understood/mechanisms appear to differ
no mt tRNAs encoded by mt
RIC ytRNA--gt repair mt defect in human cell line
Mahata et al Science (2006) 314471
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RNA import into mitochondria
poorly understood/mechanisms appear to differ
all but one tRNA encoded on mt genome
import depends on protein import
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Specialized import into mitochondria
Piggyback on tRNA import
proteinRNA conjugate
Bind to mtRNA binding protein
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Unreal Irrational
Undergraduate teaching with SAGA deletions
Regulating RNA degradation in yeast mitochondria
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Expression Engineering Experiment
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
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FY2068 A ura3-52 his3?200 leu2?1 lys2-128d
Subunit Deleted?
ADA3 1/1
GCN5 1/1
SPT3 (3 groups) 3/3
SPT8 (3 groups) 0/3
UBP8 (2 groups) 2/2
SUS1 (2 groups) 2/2
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FY2068 A ura3-52 his3?200 leu2?1 lys2-128d
Subunit Deleted?
ADA3 1/1
GCN5 1/1
SPT3 (3 groups) 3/3
SPT8 (3 groups) 0/3
UBP8 (2 groups) 2/2
SUS1 (2 groups) 2/2
NY389 a ura3-52 his4-917d leu3?1 trp1-63
spt8?320LEU2
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Day 3
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Follow-up with microarray
wt/sgf73?
teacher
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Follow-up with spot tests
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Follow-up with spot tests
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.
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Sus1s role in SAGA-dependent gene motility,
transcription, and expression under different
cellular conditions
Andrew Ji and Kate Broadbent
May 10, 2007
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From Neal Lerner ltnlerner_at_MIT.EDUgt Subject Re
109 writing assignment Date Thu, 11 Jan 2007
110734 -0500 To natalie kuldell
ltnkuldell_at_mit.edugt 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 20.109), we have a shot at
it. See you on the 22nd. Neal
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the end
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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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Chromatin obstacle to all DNA-templated processes
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