Figure 2. ERAL1 is required for assembly of the 28S small mitoribosomal subunit.

(A) Western blot of HEK-293T cell lysate (5, 10 or 15 μg) pretreated with NT or three independent ERAL1-targeted siRNAs for 3 days and probed with anti-ERAL1 or anti-β-actin antibodies. UTR, siRNA targeting the the 3′-UTR of Eral1; ORF1 and ORF2, siRNAs targeting regions of the open reading frame. In all further siRNA experiments, depletion of ERAL1 was confirmed by Western blotting. (B) Western blot of immunoprecipitate from cells expressing MRPS26–FLAG following 3 days of non-targeting (NT) or ERAL1-depletion (ORF1) siRNA. Position of mitoribosomal subunits are indicated with the markers DAP3, MRPS18B and MRPS25 for the28S mt-SSU, and MRPL3 and MRPL12 for the 39S mt-LSU. Quantification was performed on three independent experiments. On each occasion, signals were normalized to levels of MRPS26 in the immunoprecipitate. Results are means+S.D. (C) Western blot of immunoprecipitate from cells expressing ICT1–FLAG. Experimental details and data analysis as described for panel (A) (n=3). (D) Northern blot of 4 μg of RNA isolated from HEK-293T cells treated with si-NT or following ERAL1 depletion (si-ORF1) for 4 days. RNA from four independent experiments is shown. Probes highlight transcripts encoding components of complex I (MTND2), complex III (MTCYB; mitochondrially encoded cytochrome b) and complex IV (MTCO3, COX3) as well as both 16S (MT-RNR2) and 12S (MT-RNR1) mt-rRNAs. A probe to human 18S rRNA is shown as a loading and quality control (18S). The quantification is shown for 14 independent transcripts from four repeats. Results are means+S.D. ***P<0.001, **P<0.01, *P<0.05.