The zinc-binding domain of mammalian prolyl-tRNA synthetase is indispensable for catalytic activity and organism viability

Vasu, K.I.; Ramachandiran, Iyappan; Terenzi, Fulvia; Khan, Debjit; China, Arnab; Khan, Krishnendu; Chechi, Aayushi; Baleanu-Gogonea, Camelia; Fox, Paul L.

doi:10.1016/j.isci.2021.102215

Cited by 4 publications

(8 citation statements)

References 46 publications

(40 reference statements)

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“…Probe design for LNA probe-based PCR requires prior knowledge of the mutation through prior sequencing in the case of NHEJ, or from knowledge of the repair template used for HDR. These approaches have been used to successfully identify and breed mice generated by CRISPR-Cas9 with a point mutation in Eprs that deletes a portion of the zinc-binding domain ( Vasu et al., 2021 ). Note: This protocol can also be adapted to other species or cell lines.…”

Section: Before You Beginmentioning

confidence: 99%

“…LNA bases are underlined (blue). Figure reprinted with permission from Vasu et al., 2021 . CRITICAL: Fluorophore dyes compatible with the specific qPCR instrument should be selected.…”

Section: Before You Beginmentioning

confidence: 99%

“… (B) qPCR curves showing amplification and selective detection using wt (left) and mut (right) probe sets. Figure reprinted with permission from Vasu et al., 2021 . …”

Section: Expected Outcomesmentioning

confidence: 99%

“…Locked nucleic acid probe-based qPCR provides rapid identification of the mutant allele and can be used for genotyping offspring during subsequent breeding for colony establishment. For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021) .…”

mentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021) .…”

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confidence: 99%

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Screening of CRISPR-Cas9-generated point mutant mice using MiSeq and locked nucleic acid probe PCR

Vasu

Fox

2021

STAR Protocols

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Summary CRISPR-Cas9-mediated, site-directed mutagenesis in mice generates mosaic founder mice with varied efficiency of desired point mutation and other non-homologous end-joined variants. Here, we present a protocol for design, sample preparation, and analysis for identification of mice with the desired mutation. Deep sequencing provides the proportion of reads of a particular allele for each mouse line. Locked nucleic acid probe-based qPCR provides rapid identification of the mutant allele and can be used for genotyping offspring during subsequent breeding for colony establishment. For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021) .

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Section: Before You Beginmentioning

confidence: 99%

“…LNA bases are underlined (blue). Figure reprinted with permission from Vasu et al., 2021 . CRITICAL: Fluorophore dyes compatible with the specific qPCR instrument should be selected.…”

Section: Before You Beginmentioning

confidence: 99%

“… (B) qPCR curves showing amplification and selective detection using wt (left) and mut (right) probe sets. Figure reprinted with permission from Vasu et al., 2021 . …”

Section: Expected Outcomesmentioning

confidence: 99%

mentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Vasu et al. (2021) .…”

mentioning

confidence: 99%

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Screening of CRISPR-Cas9-generated point mutant mice using MiSeq and locked nucleic acid probe PCR

Vasu

Fox

2021

STAR Protocols

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Homozygous EPRS1 missense variant causing hypomyelinating leukodystrophy-15 alters variant-distal mRNA m6A site accessibility

Khan,

Ramachandiran,

Vasu

et al. 2024

Nat Commun

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Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.

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Cardiomyocyte-specific Loss of Glutamyl-prolyl-tRNA Synthetase Leads to Disturbed Protein Homeostasis and Dilated Cardiomyopathy

Wu,

Hollinger,

Bonanno

et al. 2023

Preprint

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Glutamyl-prolyl-tRNA synthetase (EPRS1), an aminoacyl-tRNA synthetase (ARS) ligating glutamic acid and proline to their corresponding tRNAs, plays an essential role in decoding proline codons during translation elongation. The physiological function of EPRS1 in cardiomyocytes (CMs) and the potential effects of CM-specific loss of EPRS1 remain unknown. Here, we found that heterozygous Eprs1 knockout in CMs does not cause any significant changes in CM hypertrophy induced by pressure overload, while homozygous knockout leads to dilated cardiomyopathy, heart failure, and lethality at around 1 month after Eprs1 deletion. Transcriptomic profiling of early-stage Eprs1 knockout hearts suggests a significantly decreased expression of multiple ion channel genes and an increased gene expression in proapoptotic pathways and integrated stress response. Proteomic analysis shows decreased protein expression of multi-aminoacyl-tRNA synthetase complex components, fatty acid, and branched-chain amino acid metabolic enzymes, as well as a compensatory increase in cytosolic translation machine-related proteins. Immunoblot analysis indicated that multiple proline-rich proteins were reduced at the early stage, which might contribute to cardiac dysfunction of Eprs1 knockout mice. Taken together, this study demonstrates the physiological and molecular outcome of loss-of-function of EPRS1 in vivo and provides valuable insights into the potential side effects on CMs resulting from the EPRS1-targeting therapeutic approach.

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The zinc-binding domain of mammalian prolyl-tRNA synthetase is indispensable for catalytic activity and organism viability

Cited by 4 publications

References 46 publications

Screening of CRISPR-Cas9-generated point mutant mice using MiSeq and locked nucleic acid probe PCR

Screening of CRISPR-Cas9-generated point mutant mice using MiSeq and locked nucleic acid probe PCR

Homozygous EPRS1 missense variant causing hypomyelinating leukodystrophy-15 alters variant-distal mRNA m6A site accessibility

Cardiomyocyte-specific Loss of Glutamyl-prolyl-tRNA Synthetase Leads to Disturbed Protein Homeostasis and Dilated Cardiomyopathy

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