HEATR3 variants impair nuclear import of uL18 (RPL5) and drive Diamond-Blackfan anemia

Abstract

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.

Graphical abstract

Figure 1.

Clinical presentation and HEATR3 variants that drive DBA. (A) Photographs of the face and hands of the affected P1, P2, P3, P5, and P6 (photos from Family C not available). No common facial finding was present in affected individuals, but straight eyebrows, down-slanting palpebral fissures, and synophrys are apparent in some. Note that fingers in P1, P3, and P6 appear disproportionately short compared with the hand. Also note the presence of thumb anomaly in P5. (B) The pedigrees of Families A to D with genotypes for specified HEATR3 variants are indicated below each available individual. The pathogenic variants are indicated in red. (C) HEATR3 variants cosegregating with DBA in one affected individual from each family arranged according to position of coding sequence in ascending order. The missense variants c.400T>C (p.Cys134Arg) in P5, c.1337G>A (p.Cys446Tyr) in P3, and c.1751G>A (p.Gly584Glu) in P1 and P2 are homozygous; the splice donor site variant c.399 + 1G>T (p.?) and the missense variant c.719C>T (p.Pro240Leu) are compound heterozygous in P4. (D) Schematic representation of HEATR3 with 15 exons. (E) HEATR3 protein with four ARM (Armadillo) and six HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, and Target of rapamycin 1) repeat domains, indicating the position of associated variants. The protein domains have been described previously.

Figure 2.

The HEATR3 Gly584 (yeast Gly522) residue is important for protein function. (A) Polysome profiles of wild-type and syo1Δ yeast cells expressing empty vector controls or HA-tagged Syo1-Gly522 mutations display an altered 60S/40S subunits ratio. Representative examples of a duplicate are shown. (B) Mutations at position Gly522 do not complement yeast growth. The mild growth impairment observed in syo1Δ cells is complemented by a wild-type construct (HA-Syo1) but not by constructs harboring a mutation at position Gly522. The indicated strains were grown at 16°C on synthetic medium lacking leucine for 6 days. (C) The Gly522 mutants are stably expressed in yeast. HA-Syo1 constructs were detected by western blotting with an anti-HA antibody. As loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probing was performed. (D) Position of yeast mutation Gly522 on the three-dimensional structure of C thermophilum Syo1 in complex with L5-N (based on PDB 4GMN²⁵). Yeast Gly522 corresponds to Gly584 in the C-terminal (supplemental Figure 1). Two views are shown.

Figure 3.

HEATR3 variants affect protein levels and uL18 nuclear localization. (A) Representative western blot of lysates from LCLs derived from P2 and P3 that were either untreated or exposed to the proteasome inhibitor MG-132. Specific antibodies are used to detect the relative amounts of HEATR3. Protein p53 provides a positive control showing proteasome inhibition by MG-132. Actin detection was used as loading control. (B) Real-time polymerase chain reaction analysis of complementary DNAs generated from LCLs derived from a healthy control or from P2 and P3 using primers to measure HEATR3 mRNA levels. The mRNAs of genes 36B4, ACTB, and GAPDH were used as references. Data shown are the results of 6 biological replicate experiments. (C) Western blot of lysates from HeLa cells transfected with 2 different siRNAs targeting HEATR3 (#1 and #2) or a control scrambled siRNA (SCR) probed with antibodies against HEATR3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detection was used as loading control. (D) Wide-field fluorescence microscopy of HeLa cells transfected with SCR or HEATR3 siRNAs and stained with 4′,6-diamidino-2-phenylindole (DAPI) (to label the nucleus) and antibodies against uL18, shown at ×20 magnification. The insets show enlarged pictures of the areas framed by dotted lines. The arrowheads point to stained nucleoli, which are observed in control cells but not after HEATR3 knockdown. (E) Fibroblasts derived from a healthy control or P2, stained with DAPI and antibodies against uL18 or uL5. Scale bar, 10 μm. (F) Quantification of the number of fibroblasts revealing nuclear staining of uL18.

Figure 4.

Variants in HEATR3 affect pre-rRNA processing. (A) Northern blot analysis of LCLs derived from control or affected individuals carrying HEATR3 variants. Radiolabeled probes targeting the 5′ extremity of internal transcribed spacer 1 (5′ITS1), the ITS1/5.8S junction (ITS1-5.8S), or internal transcribed spacer 2 (ITS2) were used to detect pre-rRNA precursors. Each lane was loaded with 3 µg total RNA. (B) Quantification of pre-rRNAs in LCLs derived from individuals carrying HEATR3 variants was performed by using Ratio Analysis of Multiple Precursors: product-to-precursor ratios at various processing steps are expressed as variations relative to LCLs from healthy controls. Mean values ± standard error of the mean from 3 independent experiments, aside from sample P4, for which n = 2. (C) Restoration of HEATR3 expression in LCLs from P3. LCLs from P3 and from a control individual were transduced with lentiviruses expressing either green fluorescent protein (LV-GFP) or HEATR3 (LV-HEATR3). Western blot analysis confirmed expression of HEATR3 in P3 LCLs transduced with LV-HEATR3 (upper panel). Northern blot analysis of total pre-rRNAs with probe ITS2 showed restoration of a normal pre-rRNA processing pattern. (D) Ratio Analysis of Multiple Precursors quantification of the northern blot shown in panel C. Product-to-precursor ratios in P3 LCLs at steps related to the processing of the large subunit rRNA precursors are expressed as variations relative to LCLs from the healthy control. Mean values ± standard error of the mean from 3 independent experiments.

Figure 5.

Variants in HEATR3 impair 60S ribosomal subunit accumulation. (A) Representative polysome profiles of lysates from LCLs derived from healthy individuals or patients (P2 and P4). The 40S subunit, 60S subunit, and 80S monosome are labeled. The vertical arrow highlights the reduction of 60S subunits in the samples from P2 and P4. (B) Representative polysome profiles of lysates from LCLs transduced with lentiviruses expressing HEATR3 or GFP complementary DNA. At least 3 independent experiments were performed with each cell line (panels A and B).

Figure 6.

Erythroid cell proliferation and differentiation are impaired in individuals with HEATR3 variants. (A) Bone marrow aspirates of P2 (left panels) and P5 (right panels) with May-Grünwald-Giemsa staining, demonstrating a paucity (arrows) or absence (P5) of erythroid precursors and hypolobulated megakaryocytes (lower panels). (B) Cell count of purified CD34⁺ from the peripheral blood of P2 (orange line) and a healthy control (blue line) subject to the erythroid culture assay was performed (n = 1 to minimize invasiveness). Shown are cell counts on days 5, 7, 10, and 12. (C) Flow cytometry analysis of differentiating erythroid cells from P2 and a healthy control at days 10 and 12. (D) Quantification of panel C. (E) Reverse transcription quantitative real-time polymerase chain reaction quantification of HEATR3 transcript levels in CD34⁺ cells infected with lentiviral constructs expressing shRNAs targeting HEATR3 or a scrambled control (SCR). (F) Cell numbers on days 5, 7, 10, and 12 of an erythroid culture assay after infection with the lentiviral vectors expressing HEATR3 shRNAs. (G) Western blot analysis of the cells on day 7. Blots were probed with antibodies against HEATR3, p21, eS19 (RPS19), heat shock protein 70 (HSP70), GATA1, and pro-caspase 3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. APC, allophycocyanin; EB, early basophilic erythroblasts; LB, late basophilic erythroblasts; ortho, acidophilic erythroblasts; poly, polychromatophilic erythroblasts; proE, proerythroblasts. **P < 0.002; ***P < 0.001; ****P < 0.0001.

Figure 7.

HEATR3 loss does not affect levels of p53, uL5, or uL18. (A) Western blots of LCLs exposed to either 100 nM camptothecin (CPT) for 4 hours, or 10 µM MG-132 for 6 hours, or the vehicle control, dimethyl sulfoxide (DMSO). Blots shown are probed with antibodies against p53. HEATR3 variants do not lead to p53 stabilization on their own but they do, as expected, in combination with CPT or MG-132 treatments. (B) Quantification of panel A from 3 independent experimental replicates. Results shown are the relative levels of p53/actin. (C) Western blots of LCLs exposed to 100 nM CPT for 4 hours, or the vehicle control, DMSO. Blots shown are probed with antibodies against HEATR3, p53, uL5, and uL18. (D) Quantification of panel C from 3 independent experimental replicates. Results shown are the relative levels of uL5/actin or uL18/actin.