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
What I wanted to do this summer Evaluate inducible inactivation system designed to regulate mitochondrial gene expression Examine unintended consequences.
Dec 18, 2015
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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
Biochemistry (2008) 47: 8514
Rnt1p
dsRNAse
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
mt gene +dox
mt gene
Inducible inactivation system for mitochondrial gene expression
mt gene
Inducible inactivation system for mitochondrial gene expression
Part1: mtRNT1
FY2068 MAT ura3-52 his3200 leu2 1 lys2-128
mt gene
Inducible inactivation system for mitochondrial gene expression
Part1: mtRNT1
Is mtRNT1 induced +dox?
Is mtRNT1 localized to mt?
Are there consequences of induction/localization?
anti-Rnt1 Western
1 Marker2 - mtRNT, - dox3 + mtRNT, - dox4 - mtRNT, + dox5 +mtRNT, + dox
Is mtRNT1 induced + dox?
1 2 3 4 5
“- mtRNT”
“+ mtRNT”
50 kDa
75
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
“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.”
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
DAPI
Alexa
1:4000 1° Ab
100X total magnification
MH36 (YPD)
Is mtRNT1 localized to mt?
“+ mtRNT”- dox + dox
DAPI
Alexa
DAPI
Alexa
1:4000 1° Ab
100X total magnification
DIC
+mtRNT1 -dox
DAPI
Alexa
1:4000 1° Ab
100X total magnification
+mtRNT1 -dox
DAPI
Alexa
1:4000 1° Ab
100X total magnification
DIC
+mtRNT1 +dox
DAPI
Alexa
1:4000 1° Ab
100X total magnification
+mtRNT1 +dox
Next:
Try GFP fusion to mtRNT1?
= - mtRNT +dox = + mtRNT +dox
Agilent 4x44K spotted array, 3DNA labeling
Are there consequences of induction/localization?
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
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