Results: 4

1.
FIGURE 2.

FIGURE 2. From: Global Identification of New Substrates for the Yeast Endoribonuclease, RNase Mitochondrial RNA Processing (MRP).

Genetic interaction between cleavage targets and RNase MRP. The indicated mutant yeast strains were serially diluted and replica-pinned onto either synthetic dextrose (−Ura) plates or 5-flouroorotic acid (5-FOA) plates. The cells in each of the cultures contain a plasmid with a URA3 marker and wild-type copy of NME1. Cells can grow on 5-flouroorotic acid if they no longer require a wild-type NME1. All plates were grown at 28 °C for 3–4 days. A, synthetic sick phenotype of sic1/nme1 double mutants. B, synthetic sick phenotype of nme1, U2 components, and splicing proteins.

Jason Aulds, et al. J Biol Chem. 2012 October 26;287(44):37089-37097.
2.
FIGURE 1.

FIGURE 1. From: Global Identification of New Substrates for the Yeast Endoribonuclease, RNase Mitochondrial RNA Processing (MRP).

Northern blot analysis of potential targets in wild-type and RNase MRP mutants. Total RNA from wild-type and RNase MRP mutant cells was separated on a formaldehyde-agarose denaturing gel, transferred, and probed for VPH1 mRNA (control) and either SIC1 (A), CTS1 (B), or LSR1 (C). RNA levels for each sample were normalized to the loading control VPH1 RNA and to the wild type as measured using ImageQuant 5.0 image analysis software. Wild type was given a relative value of 1.0. Calculated values are listed under each set of gels.

Jason Aulds, et al. J Biol Chem. 2012 October 26;287(44):37089-37097.
3.
FIGURE 3.

FIGURE 3. From: Global Identification of New Substrates for the Yeast Endoribonuclease, RNase Mitochondrial RNA Processing (MRP).

A, cleavage of in vitro transcribed CTS1 5′-UTR RNA and 5.8 S rRNA A3 by purified RNase MRP. 3′-End-labeled RNA of the 5.8 S rRNA A3 site was used as a positive control for RNase MRP cleavage activity. Similarly labeled CTS1 5′-UTR RNA was generated, and both were subjected to increasing amounts of RNase MRP enzyme for 30 min. B, determination of RNase MRP cleavage sites on the CTS1 5′-UTR. Cleavage products generated from in vitro transcribed CTS1 5′-UTR by purified RNase MRP were subjected to primer extension with CTS1-R3 primer and separated on a sequencing gel next to the CTS1 5′-UTR DNA sequence using the same primer. Positions of cleavage sites (*1–*9) are indicated and are mapped in supplemental Fig. 1. nt, nucleotides.

Jason Aulds, et al. J Biol Chem. 2012 October 26;287(44):37089-37097.
4.
FIGURE 4.

FIGURE 4. From: Global Identification of New Substrates for the Yeast Endoribonuclease, RNase Mitochondrial RNA Processing (MRP).

Northern blot analysis of potential RNase MRP substrates. A, Northern blot analysis of CTS1 and Ty1 levels in synchronized NME1 (wild-type) and nme1-Δ2 strains. NME1 and nme1-Δ2 strains were synchronized by treatment with hydroxyurea, released, and then sampled every 15 min as indicated. SCR1 levels were used as a loading control. Levels of CTS1 transcript were plotted and are relative to the SCR1 and zero time points. B, Northern blot analysis of CTS1 mRNA levels in wild type (WT), Δxrn1, nme1-Δ2, and Δxrn1/nme1-Δ2 strains. Total RNA from wild type and the indicated mutants was separated on a formaldehyde-agarose denaturing gel, transferred, and probed for CTS1. Methylene blue staining of 18 S rRNA was used as a loading control. The -fold change in level from the control wild-type lane is indicated below the gel.

Jason Aulds, et al. J Biol Chem. 2012 October 26;287(44):37089-37097.

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