Transcript Turn it down: tempering natural gene expression in the nucleus and

Synthetic Systems for
Teaching and Learning
Winston Retreat
June 25th, 2007
Natalie Kuldell
Unreal
Irrational
Regulating RNA
degradation in yeast
mitochondria
Undergraduate
teaching with SAGA
deletions
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/Interface2
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. Tunable
vary operation at will
Current contents: in mt from mt
mt genome includes
• 8 protein coding genes
7 oxphos, 1 riboprot
• 2 rRNAs
• 24 tRNAs
mt promoters
mt RNAP
TATAAGTA
(+1)
RPO41 = catalytic subunit
MTF1 = specificity factor
nuclearencoded
Targeted mtRNA degradation
Part 1: mRNA target
e.g. mtGFP
Part 2: guide RNA
Part 3: dsRNase
Snapshot of wild type role for Rnt1
• Localized to the nucleus even when overexpressed
Catala et al, MCB (2004) 15:3015
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
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) 15:140
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) 15:3015
Expression vector for mitochondrial Rnt1
CMV
CYC1
modified RNT1
tTA
2x tetO
pRS41n
Expression vector for mitochondrial Rnt1
signal sequence
+ epitope tag
RNT1
∆NLS
(11 aa)
pRS41n
∆NLS in Henras et al RNA (2004) 10:1572
Initial experiments with mtRnt1
1. Expression? by Western with epitope Ab
2. Phenotypes? Respiration, growth, existing markers
3. Overall? Microarray wt vs mtRnt
Targeted mtRNA degradation
Part 1: mRNA target
e.g. mtGFP
Part 2: guide RNA
Part 3: dsRNase
Protein import into mitochondria
Pfanner and
Geissler Nat Rev
(2001) 2:339
RNA import into mitochondria
“poorly understood”/”mechanisms appear to differ”
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
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
RNA import into mitochondria
“poorly understood”/”mechanisms appear to differ”
all but one tRNA
encoded on mt
genome
import depends
on protein import
Specialized import into mitochondria
protein:RNA
conjugate
Piggyback
on tRNA
import
Bind to
mtRNA
binding
protein
Unreal
Irrational
Regulating RNA
degradation in yeast
mitochondria
Undergraduate
teaching with SAGA
deletions
Expression Engineering Experiment
Day 1
Day 2
Day 3
RT
Day 6
Day 5
Day 4
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
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∆320::LEU2
Day 3
Follow-up with microarray
wt/sgf73∆
wt/sus1∆
teacher
Andrew Ji and Kate Broadbent,
W/F Team Blue, 20.109 Spring ‘07
Follow-up with spot tests
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.
Sus1’s role in SAGA-dependent gene
motility, transcription, and expression
under different cellular conditions
Andrew Ji and Kate Broadbent
May 10, 2007
From: Neal Lerner <[email protected]>
Subject: Re: 109 writing assignment
Date: Thu, 11 Jan 2007 11:07:34 -0500
To: natalie kuldell <[email protected]>
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
the end
Current contents: in mt from nucleus
nuclear genome sends
• ~750 proteins to mt
87 of these are putative
proteins of no known function