Disrupted Expression of the Splicing Regulators Tra2-Beta and SRSF1 and Subsequent Aberrant Splicing of FLVCR1 Transcript As an Underlying Mechanism for the Erythropoietic Failure Observed in Diamond-Blackfan Anemia
Bibliographic record
Abstract
Abstract Diamond Blackfan Anemia (DBA) is a rare inherited anemia caused by disruption of erythroid progenitor cell development at the burst-forming and colony-forming units. Approximately 50% of DBA patients have additional non-hematopoietic abnormalities including heart defects and short stature. Sixteen genes encoding specific ribosomal proteins (RPs) and the transcription factor GATA1 have been found mutated in approximately 65% of DBA patients, with the RPS19 gene being mutated in 25% of patients. Because RPs are components of the ribosome that is critical for protein synthesis, the identification of RP mutations suggest a translation deficiency as a mechanism for erythroid progenitor failure. However, erythropoietic failure in DBA may arise by causes other non-translational functions of the DBA genes. We have previously reported that inhibiting the heme exporter protein FLVCR1 in human hematopoietic stem cells and progenitors (HSC/Ps), is sufficient to specifically block erythropoiesis, which mimics the hematological features observed in DBA. Moreover, we reported aberrant splicing of exons (E) 2 and 3 of the FLVCR1 transcript in DBA erythroid cells that resulted in disruption of FLVCR1 protein expression and function. We observed aberrant FLVCR1 splicing in erythroid cells from patients with RPS19 and RPL5 mutations and from typical DBA patients whose genetic background has not been known at that time; thus, suggesting FLVCR1 dysfunction as central for the DBA erythropoietic failure. In the present work, we obtained evidence suggesting that DBA pathogenesis is caused by abnormality in the cellular splicing machinery. We performed In silico analysis of the binding motifs of seven SR proteins on each of the FLVCR1 exons. We found binding motifs for TRA2-β, SRSF1 and 9G8 on E2 and/or E3, the two FLVCR1 exons that are skipped in DBA erythroid cells and in RPS19-disrupted K562 cells. We focused on expression analysis of TRA2B and SRSF1 in RPS19-reduced K562 cells. Expression of both TRA2B and SRSF1 mRNA were significantly disrupted in RPS19-disrupted K562 cells, which correlated with disrupted expression of their respective proteins. To test the specificity of disrupted SR splicing regulators and aberrant FLVCR1 splicing, we analyzed TRA2-β, and SRSF1 protein expression, and FLVCR1 aberrant splicing, in K562 cells disrupted in SBDS gene. The SBDS gene product is critical for ribosome biogenesis and the gene is mutated in 90% of patients with Shwachman-Diamond Syndrome. Interestingly, K562 cells down-regulated in SBDS showed normal levels of TRA2-β and SRSF1 expression, and no FLVCR1 aberrant splicing was observed. Importantly, erythroid differentiation potential of the DBA cell model can be rescued by expression of exogenous Tra2-β cDNA or FLVCR1 cDNA. Altogether our data suggests that mutations in the ribosome protein genes in DBA result in down-regulation of the splicing factors, TRA2-β and SRSF1, which impair normal FLVCR1 splicing and consequently erythropoiesis failure. Disclosures No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".