Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.
Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.
Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.
The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how tunicamycin treatment, that induces eIF2 phosphorylation, affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict the sensitivity of transcripts to altered ribosome recruitment in presence or absence of Tm. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.
The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how eIF2 phosphorylation affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict eIF2-dependent translation efficiency, but uORF identity differs. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.- eif2 dependent translation in erythroblasts during proteotoxic stress determined by ribosome footprinting- identification of transcription factors upregulated in response to eIF2 phosphorylation- Advantages and disadvantages of translation initiation site determination using harringtonine- distinct uORF pattern in transcripts with enhanced, or more than average reduced translation upon proteotoxic stress
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