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