Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy

Abstract

The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/ threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively, this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoforms we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β proteins encoded by independent vertebrate genes. Our analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions.

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

“…Embryos from the THattP40 ( y 1 sc v 1 sev 21 ; P{y +t7.7 v +t1.8 nos-Cas9.R}attP40 ) or THattP2 ( y 1 sc v 1 sev 21 ; P{y +t7.7 v +t1.8 nos-Cas9.R}attP2 ) lines expressing nos -Cas9 were injected using standard procedures (Bloomington Drosophila Stock Centre). Donor DNA (500 ng/μL) in sterile H 2 O was injected together with gRNA plasmids (100 ng/μL) as described previously ( Korona et al, 2020 ). Individually selected surviving adults were crossed to w 1118 and the progeny screened for DsRED fluorescence localised mostly to the eyes of transgenic flies: positive flies were balanced and homozygous stocks established where possible.…”

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

“…Embryos were injected using standard procedures into the THattP40 or THattP2 lines expressing nos-Cas9. Donor DNA (500 ng/μL) in sterile dH2O was injected together with of gRNA plasmids (100 ng/μL) as described previously 47 . Individually selected surviving adults were crossed to w 1118 and the progeny screened for DsRED fluorescence localized mostly to the eyes of transgenic flies: positive flies were balanced and homozygous stocks established where possible.…”

“…For generation of donor vectors, firstly, homology arms were amplified on genomic DNA (Supplementary Table 4) that, secondly, were used as a template to amplify the homology arms (Supplementary Table 5) of the donor vector for CRISPR/Cas9 homologous recombination (HDR). The inserts with visible marker were amplified using as a template previously generated constructs 47 with appropriate primers. These fragments were used for Gibson assembly using Gibson Assembly Master Mix (New England Biolabs).…”

“…For tagging of Dα6 nAChRs subunit the C-terminal fusion with FSVS fluorescent protein harbouring StrepII and 3xFLAG epitope tags (3xFLAG-StrepII-Venus-StrepII) was generated for CRISPR/Cas9 mediated genome engineering 47,48 . Firstly, gRNAs were designed (Supplementary Table 3) and tested against the genomic DNA sequence of injection strains.…”

“…In order to generate individual nAChR subunits gene deletions the open reading frame (ORF) was disrupted by introducing a visible marker harbouring DsRED marker under eye specific driver 3Px3 using CRISPR/Cas9 technology as previously described 47 . The targeted exons are shared between different isoforms and adjacent to the N-terminus to ensure the protein translated was interrupted.…”