Bone Marrow Failure and Developmental Delay Caused By Mutations in Poly(A)-Specific Ribonuclease

Background: Deadenylation is a major mechanism that regulates RNA function and fate. Several mammalian deadenylases have been identified. Poly (A)-specific ribonuclease (PARN) is one of the major mammalian deadenylases that trims single-stranded poly (A) tails of mRNAs and oligoadenylated tails of H/ACA box snoRNAs and microRNAs. RNA biogenesis has emerged as a mechanism underlying several inherited diseases including well known inherited bone marrow failure syndromes (IBMFSs) such as Diamond Blackfan anemia, dyskeratosis congenita and Shwachman-Diamond syndrome. Little is known about the biological significance of germline mutations in PARN. Methods: Genome-wide screen for copy number alterations was used to identify causal mutations in patients with hematological and neurological manifestations. Four patients were identified with deletions in the PARN gene. Genomic, biochemical, cellular and knockdown experiments in human bone marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. Results: We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination, and severe bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and had severely reduced PARN protein and deadenylation activity. Clonogenic assays of the patient's bone marrow cells showed reduced potential to generate hematopoietic colony. Fibroblasts from the patient with biallelic mutations showed markedly slow growth. Large proportion of the cells accumulated in the G1/G0 phase of cell cycle, suggesting impaired transition from G1 to the S phase. Cells from this patient also had impaired oligoadenylation of specific H/ACA box snoRNAs and scaRNAs, including the telomerase RNA component (TERC). Importantly, PARN-deficient patient cells manifested abnormalities in two pathways that are affected in IBMFSs: short telomeres and an aberrant ribosome profile. This combination of abnormalities is seen in patients with severe variants of dyskeratosis congenita (Hoyeraal-Hreidarsson syndrome). Knocking down PARN in human CD34+ marrow cells from healthy donors revealed marked defect in clonogenic potential. Morpholino knockdown of parn in zebrafish resulted in reduced formation of red blood cells and granulocytes. Conclusions: We report for the first time four patients from three different families with developmental delay or mental illness, who carried large monoallelic PARN deletions that have not been reported in healthy controls. This indicates that large PARN deletions in humans result in a neurological phenotype. Further, we showed that biallelic PARN mutations that results in markedly reduced protein, cause severe bone marrow failure and severe global central hypomyelination, similar to what is seen in patients with severe forms of dyskeratosis congenita. The identified defects suggest a new disease mechanism, in which PARN-deficiency disrupts the polyadenylated state of H/ACA box RNA molecules that in turn influences ribosome profile and telomere length and cause hematological and neurological defects. Disclosures No relevant conflicts of interest to declare.

Growth Phase-Coupled Changes of the Ribosome Profile in Natural Isolates and Laboratory Strains of Escherichia coli

ABSTRACT The growth phase-dependent change in sucrose density gradient centrifugation patterns of ribosomes was analyzed for both laboratory strains of Escherichia coli and natural isolates from the ECOR collection. All of the natural isolates examined formed 100S ribosome dimers in the stationary phase, and ribosome modulation factor (RMF) was associated with the ribosome dimers in the ECOR strains as in the laboratory strain W3110. The ribosome profile (70S monomers versus 100S dimers) follows a defined pattern over time during lengthy culture in both the laboratory strains and natural isolates. There are four discrete stages: (i) formation of 100S dimers in the early stationary phase; (ii) transient decrease in the dimer level; (iii) return of dimers to the maximum level; and (iv) dissociation of 100S dimers into 70S ribosomes, which are quickly degraded into subassemblies. The total time for this cycle of ribosome profile change, however, varied from strain to strain, resulting in apparent differences in the ribosome profiles when observed at a fixed time point. A correlation was noted in all strains between the decay of 100S ribosomes and the subsequent loss of cell viability. Two types of E. coli mutants defective in ribosome dimerization were identified, both of which were unable to survive for a prolonged period in stationary phase. The W3110 mutant, with a disrupted rmf gene, has a defect in ribosome dimerization because of lack of RMF, while strain Q13 is unable to form ribosome dimers due to a ribosomal defect in binding RMF.