RNA exosome mutations in pontocerebellar hypoplasia alter ribosome biogenesis and p53 levels

Müller, Juliane S.; Burns, D T; Griffin, Helen; Wells, Graeme Raymond; Zendah, Romance A; Munro, Benjamin; Schneider, Claudia; Horvath, Rita

doi:10.26508/lsa.202000678

Cited by 23 publications

(23 citation statements)

References 86 publications

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“…Cerebellar atrophy and microcephaly were observed in exosc8 and exosc9 mutant zebrafish with significant increase in the transcript levels of p53. Increased p53 correlates with increased number of brain cells undergoing apoptosis during development 17 . These findings reflect phenotypes observed in the patients with variants in genes coding for exosome subunits and the importance of exosome complex in brain development.…”

Section: Discussionsupporting

confidence: 54%

“…This finding suggests that change in the amino acid from serine to leucine at p.35 might affect the stability of the EXOSC1 protein. Previously, missense variants in different exosome subunits were shown to significantly reduce the amount of protein 9,10,17 . Each subunit of the complex is critical for its assembly and defects in any one subunit might affect the complex formation 10 .…”

Section: Discussionmentioning

confidence: 99%

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Bi‐allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia

et al. 2021

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RNA exosome is a highly conserved ribonuclease complex essential for RNA processing and degradation. Bi‐allelic variants in exosome subunits EXOSC3, EXOSC8 and EXOSC9 have been reported to cause pontocerebellar hypoplasia type 1B, type 1C and type 1D, respectively, while those in EXOSC2 cause short stature, hearing loss, retinitis pigmentosa and distinctive facies. We ascertained an 8‐months‐old male with developmental delay, microcephaly, subtle dysmorphism and hypotonia. Pontocerebellar hypoplasia and delayed myelination were noted on neuroimaging. A similarly affected elder sibling succumbed at the age of 4‐years 6‐months. Chromosomal microarray returned normal results. Exome sequencing revealed a homozygous missense variant, c.104C > T p.(Ser35Leu) in EXOSC1 (NM_016046.5) as the possible candidate. In silico mutagenesis revealed loss of a polar contact with neighboring Leu37 residue. Quantitative real‐time PCR indicated no appreciable differences in EXOSC1 transcript levels. Immunoblotting and blue native PAGE revealed reduction in the EXOSC1 protein levels and EXO9 complex in the proband, respectively. We herein report an individual with the bi‐allelic variant c.104C>T p.(Ser35Leu) in EXOSC1 and clinical features of pontocerebellar hypoplasia type 1. Immunoblotting and blue native PAGE provide evidence for the pathogenicity of the variant. Thus, we propose EXOSC1 as a novel candidate gene for pontocerebellar hypoplasia.

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Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

Bi‐allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia

et al. 2021

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“…Pre-rRNA processing and modification factors comprised a sizeable subset of factors with significant 5-EU mean percent inhibition. In total, 19/58 factors that inhibited nucleolar rRNA biogenesis were critical for processing, including the t-UTPs HEATR1/UTP10, NOL11/UTP8, and UTP4 (Gallagher et al, 2004; Prieto and McStay, 2007; Freed and Baserga, 2010; Freed et al, 2012; Turi et al, 2018), the C/D box snoRNP scaffolds NOP56 and NOP58 (Watkins and Bohnsack, 2012), as well as other processing factors including DNTTIP2 (Tafforeau et al, 2013), WBP11 (Carbon and Mungall, 2021), MDN1 (Raman et al, 2016), ESF1 (Tafforeau et al, 2013; Chen et al, 2018), BCCIP (Ye et al, 2020), RPP30 (Stolc and Altman, 1997; Tafforeau et al, 2013), EXOSC9 (Muller et al, 2020), NUMA1 (Farley-Barnes et al, 2018), MPHOSPH10 (Westendorf et al, 1998), TRMT112 (Zorbas et al, 2015), UTP20 (Wang et al, 2007), and NUDT16 (Ghosh et al, 2004). In addition, nucleolar rRNA biogenesis was moderately inhibited for the pre-rRNA modification factors TRMT112 (Zorbas et al, 2015), RPUSD2 (Carbon and Mungall, 2021), and NOLC1 (Yang et al, 2000; Werner et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Bryant

McCool

Abriola

et al. 2021

Preprint

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Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis, and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting ribosome biogenesis. However, there is a need for a more direct high-throughput assay for nucleolar function to further evaluate hits. Previous reports have monitored nucleolar RNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay for nucleolar function which enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay utilizes two siRNA controls, a negative non-targeting siRNA control (siNT) and a positive siRNA control targeting POLR1A (siPOLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dissociation. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of ribosome biogenesis in health and disease.

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“…Images were obtained using a Leica M205FA stereomicroscope (Leica Microsystem, WetzlarGermany). The body length and eye size of 120 hpf larvae were measured using ImageJ 64 software [78]. Images of the hindbrain area were acquired using the Tg(tagRFP-T:PC:GCaMP5G) stable transgenic line, and the area in µm 2 of the region of interest (ROI) was calculated using ImageJ 64 software.…”

Section: Analysis Of Larval Morphologymentioning

confidence: 99%

“…Zebrafish embryos were incubated with 10 µg/mL acridine orange solution for 15 min in the dark; the larvae were then washed three times with E3 medium. At 48 hpf, we counted acridine orange-positive cells within a pre-defined area and a quantitative analysis was performed as described elsewhere [78,82].…”

Section: Detection Of Apoptotic Cellsmentioning

confidence: 99%

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

Naef

Marchese

Ogi

et al. 2021

IJMS

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Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

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RNA exosome mutations in pontocerebellar hypoplasia alter ribosome biogenesis and p53 levels

Cited by 23 publications

References 86 publications

Bi‐allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia

Bi‐allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

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