User:Nkuldell/mtDNA pt3

Overall goal

Make a tool to regulate gene expression of any mt gene. Tool would have two-parts and be nuclear-encoded.

• Part 1: Rnt1p (RNaseIII enzyme), targetted to mt using sig sequence from HEM1 or COX4.
• Part 2: Guide RNAs, targetted to mt using lysing-tRNA-CUU (tRK1) import system.

Foundational info: Summary of natural transcriptional control elements Biswas in PNAS 1990 87:9338 and Biswas and Getz in J Biol Chem 1990 265:19053
Foundational info: works for degradation of mRNAs in S. cerevisiae nucleus Lamontagne and Elela in PLoS One 2007 5:e472

Part 1: Rnt1p

Rnt1p dsRBD in complex with snR47 RNA hairpinFeigon PNAS 1997

Gen'l info about RNases families

From Current Opinion in Structural Biology 2007 17:77 by James M Berger and Christoph W Müller "A particularly interesting family of ribonucleases that specifically cleave double-stranded RNA serves as the topic of the review by MacRae and Doudna. The RNase III group of RNA-processing enzymes currently attracts broad attention, because two family members, Dicer and Drosha, are responsible for processing RNA transcripts into microRNA (miRNAs) and short interfering RNAs (siRNAs). RNase III proteins are often multifunctional or multisubunit assemblies, and can be classified based on domain composition. Class I RNase III enzymes function as dimers, in which the RNase domains also act as dimerization domains, whereas class II and III family members are monomeric, forming a functional RNase from the internal fusion of two class I RNase III monomers. Comparing RNase III enzymes across a wide range of species leads the authors to conclude that RNase III enzymes use accessory domains as determinants of substrate specificity. For Dicer and Drosha, these accessory domains are the PAZ domain and the additional DGCR8 protein, respectively. Substrate specificity and catalytic domains are spatially separated and, in some instances, it appears that the RNase can precisely measure the distance between the RNA recognition and cleavage sites by using an internal scaffold element that functions as a molecular ruler. Given the number of different types of small RNAs and their importance in gene regulation and other cellular processes, there are sure to be many fundamental insights that will arise from the continued study of this essential protein family."

S. cerevisiae RNases/nucleases

N-term Rnt1p struct. from EMBO 2004

• NUC1 from SGD: "major mitochondrial nuclease, has RNAse and DNA endo- and exonucleolytic activities; has roles in mitochondrial recombination, apoptosis and maintenance of polyploidy"
• Bacterial endonucleases (EcoRI, BamHI and PstI) can be expressed from the yeast nuclear genome and sent to mt

Rnt1p

• seq/struc link

• Like bacterial RNaseIII, Rnt1p has two distinct domains and functions: N-terminal nuclease domain and C-terminal dsRBD as well as non-bacterial kind of N-term extension for efficiency
  

• EMBO 2004, NMR and x-ray xtal structure of dsRBD with N-term extension

• RNT1 is not essential but null shows defects in cell cycle progression and cell morphology. This paper also demonstrates Rnt1p localization to the nucleus even when overexpressed.

• processes 3' end of noncoding RNAs , e.g. in Henras et al. RNA (2004) 10: 1572

• rnt1 and ts rnt1 strain were examined by microarray to look for coding mRNAs that might be affected by mutation and the glucose-sensitive repressor Mig2 was found to be upregulated in the mutants

Part 2: Guide RNAs

RNAs that can be cleaved by Rnt1p in vitroPLoS one 2007

Structural requirements for guide RNAs

In vitro requirements shown in PLoS one 2007

Cell components needed for moving guide RNA to mt

• piggy back on existing import system
• lysine tRNA CUU (tRK1) encoded by nuclear genome and delivered to mitochondrial matrix described almost exclusively in the work of Ivan Tarassov. Other lysine tRNAs are nuclear encoded-tRK2 which decodes UUU as lysine in the cytoplasm and mitochondrially encoded-tRK3 which decodes UUU for lysine in the mitochondria.
• The mitochondrial proteome includes ~750 proteins from nuclear genome, approx 1/2 (320/750) have mitochondrial pre-sequences and ~1/3 (225/750) have predicted transmembrane domains

Experimental checkpoints for regulated expression

Part 1: Rnt1p in mt

• regulatable promoter driving second copy of Rnt1p for mt.
  • might use GalS promoter which is undetectable in glucose, leaky in YPEG and induced in Gal. Alternatively might want doxycyline, with minimal physiological consequence to regulation by tet. "off" may not be as tight so will need to check uninduced/induced by Northern.
• add tag to gene to follow localization of protein product
• microarray induced/uninduced to look for effect of RNase in mt.
  • Note: mt directed Barnase leads to resp- cells when expressed at low level (YPEG from GALS promoter on pMT416GalS) and is toxic at high levels (SC-U/Gal), unless simultanously express mtBARSTAR inhibitor protein ("BARSTM") Mireau, Arnal and Fox in Mol Gen Genomics 2003 270:1-8. Barnase is ssRNA ribonuclease whereas Rnt1p is dsRNA directed, and structure not seq dep.
• delete C-term nuclear localization signal on mt-directed copy

Part 2: guide RNAs in mt

• Current method for yeast mt isolation