Transcript ppt - OpenWetWare

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
Nuclear gene inactivation: general framework
Nuclear gene inactivation: RNA processing
U2 snRNA
Nuclear gene inactivation: RNA processing
Rnt1p
dsRNAse
Biochemistry (2008) 47: 8514
Inducible gene inactivation: RNA folding
Inducible gene inactivation: using guide RNAs
“Short RNA guides cleavage by eukaryotic RNase III.”
PLoS ONE. (2007) 2(5):e472
Inducible inactivation system
for mitochondrial gene
expression
mt gene
+dox

mt gene
Inducible inactivation system
for mitochondrial gene
expression
Part1: mtRNT1
mt gene
FY2068
MAT ura3-52 his3200 leu2 1 lys2-128 
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

Is mtRNT1 induced + dox?
anti-Rnt1 Western
1
2
3
4 5
“- mtRNT”
75
50
kDa
“+ mtRNT”
1 Marker
2 - mtRNT, - dox
3 + mtRNT, - dox
4 - mtRNT, + dox
5 +mtRNT, + dox
 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
 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
MH36
(YPD)
DAPI
Alexa
100X total
magnification
1:4000 1° Ab
 Is mtRNT1 localized to mt?
“+ mtRNT”
- dox
DAPI
Alexa
+ dox
+mtRNT1 -dox
DAPI
DIC
100X total
magnification
1:4000 1° Ab
Alexa
+mtRNT1 -dox
DAPI
Alexa
100X total
magnification
1:4000 1° Ab
+mtRNT1 +dox
DAPI
DIC
100X total
magnification
1:4000 1° Ab
Alexa
+mtRNT1 +dox
DAPI
Next:
Try GFP
fusion to
mtRNT1?
Alexa
100X total
magnification
1:4000 1° Ab
 Are there consequences of induction/localization?
= - mtRNT +dox
= + mtRNT +dox
Agilent 4x44K spotted array, 3DNA labeling
mtRNT: most affected genes, pt 1
higher no mtRNT
I-SceII, encoded
by intron within
the mitochondrial
COX1 gene
mfold.bioinfo.rpi.edu/
mtRNT: most affected genes, pt 1
higher no mtRNT
Dubious ORF in
mt genome
within RPM1
gene
mfold.bioinfo.rpi.edu/
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)
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?
mtRNT: most affected genes, pt 2
higher + mtRNT
RNT1 upregulated
mtRNT: most affected genes, pt 2
higher + mtRNT
RNA-binding protein
End of first
part