Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome

Abstract

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5′ splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

NOC1 is a Direct MYC Target, and Its Protein Interactome Dissects Its Activity in Controlling Nucleolar Function

“…Thus, overexpression or downregulation of CEBPz in humans may also affect RNA processing, leading to defective translation. In support of this idea, the human gene rbm28, which we found in the NOC1 interactome, is responsible for the ribosomapathy-ane syndrome (Bryant et al, 2021), a rare genetic disorder caused by aberrant splicing in RBM28 pre-mRNA. This, together with other indirect information on the potential role of NOC1/CEBPz in controlling alternative splicing, highlights the potential role of the human counterpart in the control of nucleolar processes that may cause genetic disorders.…”

“…Furthermore, our study also highlights NOC1 interaction with proteins relevant for RNA processing, modification, and splicing. Indeed, we found highly represented Ythdc1 and Flacc (Fl(2)d-associated protein) and spenito (nito), the flies homolog of the nucleolar large ribosomal subunit (60S) assembly factor RBM28 (Bryant et al, 2021). Notably, all these proteins are part of the mechanism that mediates N6-methyladenosine (m6A) methylation of mRNAs (Shi et al, 2021;Deng et al, 2023).…”

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

“…While RSL24D1 and PeBoW gene mutations are not yet known to be implicated in the molecular pathogenesis of ribosomopathies, the diseases of making ribosomes, several were shown to be linked to mutations in genes encoding LSU biogenesis factors. For example, mutations in SBDS are associated with Schwachman-Diamond syndrome (Boocock et al 2003) and defects in RBM28 cause alopecia, neurological defects, and endocrinopathy (ANE) syndrome (Nousbeck et al 2008;McCann et al 2016;Bryant et al 2021). Intriguingly, RSL24D1 was recently reported to be required for murine embryonic stem cell proliferation (Durand et al 2021) and BOP1 in Xenopus anterior development (Gartner et al 2022).…”

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

“…While RSL24D1 gene mutations are not yet known to be implicated in the molecular pathogenesis of ribosomopathies, the diseases of making ribosomes, several were shown to be linked to mutations in genes encoding LSU biogenesis factors. For example, mutations in SBDS are associated with Schwachman-Diamond syndrome (Boocock et al 2003) and defects in RBM28 cause alopecia, neurological defects, and endocrinopathy (ANE) syndrome (Nousbeck et al 2008; McCann et al 2016; Bryant et al 2021). Similarly, there are several diseases caused by mutations in ribosomal proteins of the LSU including Diamond-Blackfan anemia ( RPL5; RPL11; RPL27 ) (Boria et al 2010) and X-linked intellectual disability ( RPL10 ) (Brooks et al 2014).…”