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M.W. Wlodarski et al.
compared to HeLa cells depleted of eL15.
As previously reported,23 depletion of eL15 in HeLa cells
resulted in a decrease in 32S and 12S pre-rRNAs (Figure 3A and C). This was accompanied by the accumulation of the 36S and 36S-C precursors, which are inconspicuous in normal cells, and a concomitant drop in 32.5S pre-rRNA (Figure 3B). In addition, these cells displayed lower levels of 30S pre-rRNA and higher levels of 41S and 18S-E pre- rRNAs (Figure 3A and C). This phenotype indicates that depletion of eL15 affects cleavage of the ITS1 at site 2, which promotes direct cleavage of early precursors at site E and formation of 18S-E and 36S pre-rRNAs. The 36S precursor is then trimmed by the 5’-3’ exoRNase XRN2 to produce the 36S-C and 32.5S pre-rRNAs.48 Sucrose gradi- ent analyses confirmed the efficiency of the eL15 siRNAs in reducing the free 60S subunit peak and inducing the for- mation of half-mers, as expected from the deficiency of an RPL protein (Online Supplementary Figure S3). Consistent with eL15 partial loss-of-function, an increase of 36S and
36S-C precursors was observed in the patient LCLs carry- ing the eL15 p.Tyr81* variant (Figure 3B). In addition, both cell lines displayed a marked increase of the amount of 18S-E precursors translating into a higher 18S-E/21S ratio, similar to eL15-depleted cells (Figure 3A and C). This phe- notype is similar to that previously observed in LCLs har- boring a large heterozygous deletion in RPL15.23
RPL15 mutations impair 60S ribosomal subunit formation, cell proliferation, and de novo protein synthesis
Defective processing of pre-rRNA in cells often results in cells that are unable to fully form ribosomal subunits. As such, pathogenic variants of RPs linked to DBA very often impair biogenesis of ribosomal subunits in LCLs.37 Polysome profiling was performed to determine if the observed pre-rRNA biogenesis defects resulted in impaired biogenesis of large ribosomal subunits. Figure 4A shows polysome profiles of LCL extracts derived from a
A
B
C
Figure 3. Mutations in RPL15 recapitulate specific pre-rRNA processing defects found in eL15 depleted cells. (A) Northern blot analysis of siRNA-treated HeLa cells or lymphoblastoid cell lines (LCLs) derived from individuals with Diamond-Blackfan anemia (DBA). Radiolabeled probes against ITS2 (top panel), ITS1 (middle panel), 18S or 28S (lower panel) rRNA sequences were used to blot 3μg total RNA isolated from cells. (B) Longer exposures of upper molecular weight pre-rRNA species observed in the northern blots from (A). Intensity profiles of the lanes is shown in the right-hand boxes. (C) Quantification of rRNA precursors in siRNA-treated HeLa cells (top) or LCLs (bottom) derived from individuals with DBA. The results of single experiments performed for each sample are displayed as multiple bars. Pre-rRNA ratios are normalized by dividing by the mean of the control samples.
haematologica | 2018; 103(6)