Fig. 2

H/ACA snoRTs are bound by dyskerin in the cytoplasm. A In scale schematic overview of the EIF4A1 pre-mRNA. Introns are depicted as lines connected to exons. SNORA48, SNORD10, and SNORA67 are shown. Intron-containing-SNORA67 sequence is depicted as a red line in pre-mRNA or as a red box in the EIF4A1 snoRT intron-retaining transcript. Exons flanking intron-containing-SNORA67 sequence are depicted as blue boxes. The first exon of pre-mRNA is depicted as an orange box. Diagnostic RT-qPCR amplicons are represented. The amplicon between red arrows identifies the protein coding mRNA, while the amplicon between green arrows identifies every EIF4A1 snoRTs. Primer sequences are listed in Additional file 3: Table S2. m7G: cap; AAAn: poly(A) tail; P: monophosphate. B RNA immunoprecipitation analysis of dyskerin from total cellular lysates. Top: outline of sample preparation steps. Middle: Western blot analysis of immunoprecipitated fractions from total MCF7 and MB-MDA 231 cell lysates using control IgG or anti-dyskerin antibody. A 10% input extract was used to verify the correct lysis. Bottom: RT-qPCR analysis of the known dyskerin targets (TERC, SNORA23), a known off-target (GUS), and transcripts of interest on MCF7 (left) and MDA-MB 231 (right) cell lines. Results are expressed as the fold change of dyskerin immunoprecipitation RNA level against immunoprecipitation with IgG. Data are shown as means + SEM. n=4 biological replicates were performed for each experiment. Unpaired Student’s t tests were performed on respective controls IgG. C RNA immunoprecipitation analysis of dyskerin from cytoplasmic and nuclear cell lysates. Top: outline of sample preparation steps. Centre: Western blot analysis of immunoprecipitated fractions from cytoplasmic and nuclear MCF7 cell lysates using control IgG or anti-dyskerin antibody. GAPDH was used as cytoplasmic marker, while Lamin-B1 was used as nuclear marker. A 10% input extract was used to verify the correct lysis. Bottom: RT-qPCR analysis of the known dyskerin targets (TERC, SNORA23), a known off-target (GUS), and transcripts of interest of cytoplasmic (left) and nuclear (right) RIP analysis. Results are expressed as the fold change of dyskerin immunoprecipitation RNA level against immunoprecipitation with IgG. Data are shown as means + SEM. n=4 biological replicates were performed for each experiment. Unpaired Student’s t tests were performed on respective controls IgG. EIF4A1-C *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001