Fig. 3

The dyskerin-bound EIF4A1 snoRT is truncated at its 5′ end and interacts with ribosomes in the cytoplasm. A Empirical cumulative distribution function of total cytoplasmic and polysomal-recruited transcripts. Transcripts of interest are highlighted in boxes. B RT-qPCR amplicons and relative mRNA species identified by designed primers. Exons flanking intron-containing-SNORA67 sequence are depicted as blue boxes, while the retained intron is depicted as a red box. The amplicon between green arrows identifies every EIF4A1 snoRTs (full-length EIF4A1 snoRT and 3′ EIF4A1 snoRT fragment), while the amplicon between orange arrows identifies only the full-length EIF4A1 snoRT. In purple are depicted the probes designed for digital-PCR. Primer and probe sequences are listed in Additional file 3: Table S2. C Left: Percentage representation of EIF4A1 snoRTs obtained by digital PCR absolute quantification in MCF7 cells. Data are shown as the percentage of EIF4A1 snoRT after normalization on GUS housekeeping transcript. Middle: digital PCR absolute quantification of cDNA obtained by TGIRT reverse transcriptase using oligo dT in MCF7 cells after siRNA silencing of DKC1. Data are shown as copies/microliter after normalization on GUS housekeeping transcript Right: RT-qPCR analysis of EIF4A1 snoRTs performed on samples obtained by RNA immunoprecipitation of dyskerin from MCF7 total cellular lysate, cytoplasmic and nuclear fractioning. The means from three biological replicates (n = 3) are shown; error bars represent SD. Paired Student’s t tests were performed relative to controls. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. D RT-qPCR of total RNA from MCF7 cells treated with NMDI specific inhibitors NMDI14 at 25 μM for 0, 1, 6, and 24 h (up) or with translation inhibitors cycloheximide (CHX) at 25 μg/ml for 0, 2.5, or 5 h (down). The means from three biological replicates (n = 3) are shown; error bars represent SD. Paired Student’s t tests were performed in respect of the 0 h time point. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. E Polysome profiling analysis. Top left: representative polysome profile from MCF7 cells obtained by 10–30% sucrose density gradient centrifugation. The portions of the profile referring to different ribosomal subunits are highlighted. Top right: distribution of dyskerin and control proteins across the gradient analyzed by Western blotting with specific antibodies. Bottom: distribution of transcripts of interest after RNA purification from gradient fractions obtained by RT-qPCR. Results are expressed as fraction (%) of the total amount of the transcript contained in the lysate. Data are shown as means ± SEM of three different biological replicates (N = 3). F Ribosome purification: Western blotting analysis of purified ribosomes from MCF7 cells shows a co-purification of all pseudouridine-RNP complex (DKC1, NHP2, NOP10, GAR1). RPL5 and RPS14 are shown as a positive control for ribosomes purification, while eIF4G is used as a control for ribosome-interacting factors.