Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.

Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m(7)G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.

PARN encodes poly(A)-specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal-Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN-associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa-miR-202-5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN-associated disease. K E Y W O R D S idiopathic pulmonary fibrosis, incomplete penetrance, MIR202, PARN, poly(A)-specific ribonuclease, telomere biology disorder

The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.

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.