Fig 1: Mutations in yeast DIS3 in positions analogous to those found in MM cause growth defects, molecular phenotypes and display synthetic genetic interaction with catalytic mutation in the Dis3 PIN domain. (A) Serial dilutions of indicated yeast strains were spotted on YPD plates and incubated at 25, 30 or 37°C for 60 h. V568G and A524P mutations resulted in thermosensitivity, while G833R and R847K substitutions led to growth inhibition at all tested temperatures. The two latter DIS3 point mutations seemed to give a slight synergistic effect in rrp6? background. (B) Diploid heterozygotic strains DIS3 WT/dis3-D171N G833R-pA (top) and DIS3 WT/dis3-D171N R847K-pA (bottom), combining PIN domain endo- mutation with RNB mutations analogous to those found in MM were sporulated and subjected to tetrad dissection. Only two spores from each tetrad were viable, demonstrating that MM-associated RNB domain mutations are synthetically lethal with D171N amino acid change, abolishing endonucleolytic activity of Dis3 PIN domain. (C) Total RNA isolated from the strains in (A) was subjected to northern blot analysis using probes specific to typical exosome substrates. DIS3 mutations (G833R and R847K in particular) caused accumulation of 5'-ETS, NEL025 CUT and 7S precursor of 5.8S rRNA synthesis (as well as their degradation intermediates).
Fig 2: Verification of DIS3 deletion and integration of hDIS3 expression cassette into the genome of DT40 Cre1 chicken cell line. (A) Schematic representation of the fragment of DIS3 locus in DT40 Cre1 cells before (top) and after (bottom) integration of the hDIS3 expression cassette. Exons are numbered. Positions of the left and right arm, used in recombination, and the 5-kb fragment deleted by cassette integration (removing the majority of the chicken DIS3 RNB domain from the resulting protein), as well as elements of the integration cassette are indicated. NdeI and BsaI sites used in Southern blot analysis are marked with arrows. Open arrowheads indicate LoxP sites, which were used for tamoxifen-induced Cre-mediated excision of the hDIS3 expression cassette. Horizontal light-gray bar shows location of the probe used in Southern hybridization. Lengths of the DNA fragments generated by restriction enzymes are indicated by italicized underlined numbers. Note that the size of the NdeI-derived fragment differs in the case of the locus following integration, depending on the selection marker present (Puro or Bsr). (B) Results of Southern hybridization. Total genomic DNA was isolated from the parental DT40 Cre1 cell line (lanes 2, 3, 6), heterozygotic DIS3 knockout with Puro selection marker (lanes 4, 7) or homozygotic DIS3 knockout with both Puro and Bsr selection markers (lanes 5, 8). DNA was either nondigested (lane 2) or digested with NdeI (lanes 3–5) or BsaI (lanes 6–8), transferred onto membrane and hybridized with a PCR-based probe as marked in (A). Sizes of the molecular marker (GeneRulerTM 1 kb Plus DNA Ladder from Fermentas; lane 1) are indicated on the left. Lengths of the restriction fragments recognized by the probe (indicated on the right) are as expected.
Fig 3: Substitutions of conserved amino acids in the hDIS3 RNB domain decrease enzymatic activity. (A) Schematic view of domain organization of human and yeast DIS3 proteins. Amino acids substituted in MM are indicated in light gray and their positions with respect to hDIS3 domains are marked with arrows. An asterisk indicates position of D487, which was previously shown to be critical for exoribonucleolytic activity of hDIS3. Corresponding amino acids in S. cerevisiae Dis3p are marked with italics. (B) SDS-PAGE analysis of hDIS3 protein variants purified using two-step affinity chromatography, followed by gel filtration. Position of hDIS3 protein is marked with a solid arrow. Common contaminations with bacterial DnaK chaperone and hDIS3 proteolytic degradation products are indicated with an asterisk and open arrows, respectively. The PageRulerTM Prestained Protein Ladder (Fermentas) molecular marker is in the leftmost lane. (C) TLC analysis of the RNA degradation efficiency for recombinant hDIS3 proteins, shown in (B). Internally radiolabeled RNA, synthesized by in vitro transcription in the presence of [α-32P]UTP, was incubated with equal amounts of hDIS3 variants or in the absence of protein. Aliquots were collected at indicated time points and spotted onto a PEI-cellulose TLC plate, which was developed in the direction shown with the vertical gray arrow; positions of substrate and product (UMP) are indicated with solid and open arrows, respectively. S477R, G766R and R780K mutations significantly inhibit ribonucleolytic degradation.
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