Expression of catalytically compromised DIS3 or RRP6 variants does not significantly affect rRNA synthesis and ribosome biogenesis in human cells. (A) General principle of the utilized cellular model. Plasmids compatible with Flp-In T-REx system from Invitrogen, containing wild-type or mutated variants of FLAG-tagged DIS3 or RRP6 and an EGFP-sh-miRNA fusion (both under the control of a bidirectional tetracycline-regulated promoter) were integrated into the Hek293 Flp-In T-REx cell line genome. The FLAG-tagged DIS3/RRP6 ORF was recoded in a way rendering it insusceptible to sh-miRNA silencing. Upon induction with tetracycline, stable cell lines produced either wild-type or mutated FLAG-tagged protein fusions and sh-miRNA silencing expression of only the respective endogenous gene. (B) sh-miRNA efficiently down-regulate expression of endogenous DIS3 and RRP6 at the mRNA level. Quantitative RT-PCR analysis was performed on total RNA isolated from Hek293 Flp-In T-REx cells (Ø) and established model cell lines subjected to induction with tetracycline and producing either DIS3 (WT, RNB MUT, PIN MUT, or DM) or RRP6 (WT or mut) exogenous variants simultaneously with sh-miRNAs targeting respective endogenous transcript. The graph shows results of quantification of three independent experiments. GAPDH mRNA was used for normalization. The expression level is relative to the parental Hek293 Flp-In T-REx cell line. (C) Expression of exogenous DIS3 protein variants is higher than endogenous DIS3, whereas levels of exo- and endogenous RRP6 are comparable. Model cell lines (as in panel B), or parental Hek293 Flp-In T-REx cells (control), were treated with tetracycline. Proteins extracted from cells were separated in SDS-PAGE and transferred onto nitrocellulose membranes, which were stained with Ponceau S-Red and probed with antibodies specific to FLAG epitope, EGFP, DIS3, RRP6, and β-actin (loading control). (D) Exogenous DIS3 is overexpressed around five- and 10-fold compared to endogenous protein. Western blot was performed as in panel C, but using various dilutions of the protein sample from cell line-producing DIS3 WT variant. (E) Analysis of nascent rRNA synthesis. Model cell lines cultured in a medium containing tetracycline were pulse labeled with 32P orthophosphoric acid, followed by chase in normal media for varying times (indicated above each lane). RNA was then isolated from the cells, separated in a denaturing agarose-formaldehyde gel and transferred onto nylon membrane. The blot was first stained with methylene blue (bottom part) and then subjected to phosphorimaging (upper part). Positions of 28S and 18S rRNA and other visible rRNA species are indicated on the right. In addition, the membrane was probed with hITS2a oligonucleotide (middle part) to monitor accumulation of 7S pre-rRNA, a known phenotype of DIS3 enzymatic dysfunction. Results of hybridizations with probes h5.8S and h5S (middle part) and staining of the membrane with methylene blue (bottom part) are shown to assess sample loading. (F) Selected samples from panel E were resolved in denaturing polyacrylamide gel and subsequently subjected to phosphorimaging to visualize synthesis of small RNAs (5.8S, 5S, and tRNAs) at higher resolution. (G) Ribosome/polysome profile analysis. Native cytoplasmic extracts were prepared from model cell lines, grown in the presence of tetracycline, following translation inhibition with cycloheximide, and separated by centrifugation in linear sucrose gradients. Graphs show distribution of absorbance at 254 nm from the top (left) to the bottom (right). Peaks corresponding to individual subunits (40S and 60S), monosomes (80S), and polysomes are indicated.
