Humanization of Drosophila Gαo to model GNAO1 paediatric encephalopathies

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Article

Reference

Humanization of Drosophila Gαo to model GNAO1 paediatric encephalopathies

SAVITSKIY, Mikhail, et al.

Abstract

Several hundred genes have been identified to contribute to epilepsy—the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels.

Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, we here describe the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2–3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4–7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well [...]

SAVITSKIY, Mikhail, et al . Humanization of Drosophila Gαo to model GNAO1 paediatric encephalopathies. Biomedicines , 2020, vol. 8, no. 10, p. 395

DOI : 10.3390/biomedicines8100395 PMID : 33036271

Available at:

http://archive-ouverte.unige.ch/unige:143056

Disclaimer: layout of this document may differ from the published version.

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1 D.melanogaster Gαo

C.elegans goa-1

H.sapiens GNAO1

Supplementary Figure S1. Loci of Gαo genes have variable size but similar exon-intron structures (see also Fig. 1B). NCBI Gene ID: 36104 (primary source FLYBASE:FBgn0001122) for D. melanogaster; 172505 (primary source WormBase:WBGene00001648) for C. elegans;

2775 (primary source HGNC:4389) for H. sapiens.

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2

A

dGao-PA 1 MGCAQSAEERAAAARSRLIERNLKEDGIQAAKDIKLLLLG dGao-PB 1 MGCTTSAEERAAIQRSKQIEKNLKEDGIQAAKDIKLLLLG hGao 1 MGCTLSAEERAALERSKAIEKNLKEDGISAAKDVKLLLLG ***..*******..**..**.*******.****.******

B

hGαoA 242 NRMHESLMLFDSICNNKFFIDTSIILFLNKKDLFGEKIKKSPLTICFPEYTGPNTYEDAA dGαo 242 NRMQESLKLFDSICNNKWFTDTSIILFLNKKDLFEEKIRKSPLTICFPEYTGGQEYGEAA hGαoB 242 NRMHESLKLFDSICNNKWFTDTSIILFLNKKDIFEEKIKKSPLTICFPEYTGPSAFTEAV

hGαoA 302 AYIQAQFESKNRSPNKEIYCHMTCATDTNNIQVVFDAVTDIIIANNLRGCGLY dGαo 302 AYIQAQFEAKNKSTSKEIYCHMTCATDTNNIQFVFDAVTDVIIANNLRGCGLY hGαoB 302 AYIQAQYESKNKSAHKEIYTHVTCATDTNNIQFVFDAVTDVIIAKNLRGCGLY

Supplementary Figure S2. A. Alignment of Gαo proteins (amino acids are coded by 1^st exons).

D. melanogaster has splice variants with variable 1^st coding sequence-containing exons. Most non-conservative amino acids between two Drosophila isoforms overlap non-conservative amino- acids between Drosophila and human. B. Alignment of Gαo proteins (amino acids are coded by two last exons). Human has splice variants with variable two last exons. hGαoA (settled as reference sequences here) has more identity with Drosophila’s sequences than with hGαoB (17 mismatches vs 20). There are 16 mismatches between dGαo and hGαoB.

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Supplementary Table S1. Sequence of primers used in the molecular analysis and cloning.

Primer Sequence

dGaomRNAfw GGTGAGTCGGGCAAGAGCACAATA dGaomRNArev GGCCTGGAATCCATCTTAGTACAGTCCA

LHAdGao23fw cacctgcgaatccgatTGTGGACTTTTTCAAGTGGTGGA LHAdGao23rev GAAGCCGTCCTCGTGAATGATTTTC

RHAdGao23fw gactatctttctAGGGTTAACGATTCCGCAAAATAGTAAGTACCAAATCA RHAdGao23rev atggtcttcttttcCCGGAAACTAACGCTGAGGGACGAGTG

LdGao23fw (1) CGCTGCTGCTCTTGAGTTTTCCA RdGao23rev (2) TCGTGAGTTTGCCCTTTGGCTTT

LHAdGao47fw tagtgtcttcggggccGAAAATTGAGAATGGACGGGTGGA LHAdGao47rev ttatctttctagggTTAATCTTCTCCCCGAACAAATCCT RHAdGao47fw ctatctttctagggTTAAGAAGAGTCCCCTGACGATT RHAdGao47rev atggtcttcttttcccGGTTACGGTGTTCCCTGCTAA LdGao47fw (5) AAGCTGTTTGCATAGCCAAGTGAG

RdGao47rev (6) ACAACTGTATGCAATGTTGGCGCTTGTG pUC-L (7) GCGCCTGTCACTTTGCTTGATA

pUC-R (8) CGATGGTAGTGTGGGGACTCC pBac_rev (4) GAGAGAGCAATATTTCAAGAATGC pBacWTlong (3) CCGATAAAACACATGCGTCAATTT

Lowercase letters designate overlap sequences, required for the assembly of adjacent fragments upon cloning using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). Numbers in brackets after the primers’ names represent how they are designated in Fig. 2B.