Transcripts from the src-1(cj293) mutant can encode a SRC-1 molecule lacking the SH2 domain in Caenorhabditis elegans

TL;DR

This study shows that a specific mutation in C. elegans produces a version of the SRC-1 protein missing a key domain, leading to overactivity in neurons.

Contribution

The study confirms that the src-1(cj293) mutation generates a transcript lacking the SH2 domain, supporting its role as a constitutively active allele.

Findings

RNA seq data shows the src-1(cj293) mutation leads to a splice between exon 3 and exon 6.

The resulting transcript can encode a SRC-1 molecule missing the SH2 domain.

This mutation causes overactive SRC-1 in VD neuron growth cones during outgrowth.

Abstract

Previous studies suggest that the

src-1
(
cj293
)

mutation is an activated

src-1

allele in

C. elegans

with the potential to encode a molecule lacking the SH2 domain.

src-1
(
cj293
)

is a deletion with breakpoints in introns 3 and 5, deleting exons 4 and 5, which encode the SH2 domain. If exon 3 is spliced to exon 6, the reading frame is maintained. Here, RNA seq of

src-1
(
cj293
)

mutants showed that the exon 3 to exon 6 splice does not occur in

src-1
(+)

but is robustly present in

src-1
(
cj293
)

. Thus,

src-1
(
cj293
)

produces a transcript that can encode a
SRC-1
molecular lacking the SH2 domain, which leads to overactive
SRC-1
in growth cones of VD neurons during their outgrowth (
i.e.

src-1
(
cj293
)

might be a constitutively-active mutation).

Figures

A](#f1) and B). If exon 3 is spliced to exon 6 (3-6), the reading frame is maintained, resulting in coding potential for a molecule lacking the SH2 domain and part of the kinase domain (Mahadik * et al.* 2024). RNA seq was conducted in heterozygous * [src-1](WBGene00005077) ( [cj293](WBVar00054187) )/+ * animals. Reads were aligned to the * [C. elegans](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=6239) * genome, and Sashimi plots were generated to illustrate splicing events. In animals with a * wild-type [src-1](WBGene00005077) (+) * gene, the 3-6 splice did not occur

Full text

Description

The SRC-1 /Src tyrosine kinase in * Caenorhabditis elegans * is required for embryonic development, cell migration, and axon guidance (Bei

• et al.* 2002; Itoh
• et al.* 2005; Lee
• et al.* 2005; Sugioka and Sawa 2010; Masuda
• et al.* 2012; Zhu
• et al.* 2020; Mahadik
• et al.* 2024). In developing VD axon growth cones, SRC-1 acts with the UNC-6 /Netrin receptor UNC-5 to inhibit growth cone protrusion (Mahadik
• et al.* 2024). A precise deletion mutant of

src-1 * had VD growth cones that displayed excessive protrusion resulting in axon guidance defects, similar to * unc-5 * loss-of-function (Mahadik

• et al.* 2024). The

src-1 ( cj293 ) * in-frame deletion removes exons that encode the SH2 domain and an N-terminal portion of the kinase domain (Mahadik

• et al.* 2024).

src-1 ( cj293 ) * is predicted to encode a molecule lacking the SH2 domain and part of the kinase domain (Mahadik

• et al.* 2024). Similar to the

src-1 (lq185) * precise deletion allele, * src-1 ( cj293 ) * mutants display defects in embryonic development (Bei

• et al.* 2002; Mahadik
• et al.* 2024). However,

src-1 ( cj293 ) * mutants displayed VD growth cones with reduced protrusion compared to wild-type , similar to * src-1 (+) * overexpression (Mahadik

• et al.* 2024). This suggests that

src-1 ( cj293 ) * might encode a constitutively-active SRC-1 molecule.

The breakpoints of the * src-1 ( cj293 ) * mutation are in introns 3 and 5, removing exons 4 and 5 ( Figure 1A and B). If exon 3 is spliced to exon 6 (3-6), the reading frame is maintained, resulting in coding potential for a molecule lacking the SH2 domain and part of the kinase domain (Mahadik

• et al.* 2024). RNA seq was conducted in heterozygous

src-1 ( cj293 )/+ * animals. Reads were aligned to the * C. elegans * genome, and Sashimi plots were generated to illustrate splicing events. In animals with a * wild-type src-1 (+) * gene, the 3-6 splice did not occur ( Figure 1A ). However, in * src-1 ( cj293 )/+ * animals, the 3-6 splice was common (318 times) ( Figure 1B ). The 3-6 splice products produced in * src-1 ( cj293 ) * have the potential to produce a SRC-1 molecule missing the SH2 domain and part of the kinase domain. The catalytic residue of the kinase is not in the deleted region. Kinase function is likely active in * src-1 ( cj293 ) * , as mutation of the catalytic residue (D381A) in * src-1 (syb7248) * resulted in a dominant phenotype resembling * src-1 * precise deletion, with excessively-protrusive growth cones VD growth cones (Mahadik

• et al.* 2024).

Autoinhibition of Src kinase activity is mediated by the SH2 domain, which binds to phosphorylated tyrosine 527, resulting in a closed, inactive conformation (reviewed in (Wagner

• et al.* 2013)). This tyrosine is conserved in

C. elegans * SRC-1 (tyrosine 531), thus it might also be subject to autoinhibition by the SH2 domain. A SRC-1 molecule missing the SH2 domain may result in overactive kinase activity, consistent with the * src-1 ( cj293 ) * overactive phenotype in the growth cone. It is also possible that * src-1 ( cj293 ) * overactivity is due to other interactions that require the SH2 domain and/or the N-terminal portion of the kinase domain. * src-1 ( cj293 ) * was not dominant for axon guidance defects, which would be expected of an activated molecule. Possibly, one copy of the activated allele is not sufficient to produce a phenotype in heterozygotes with * src-1 (+) * . In any case, these results are consistent with * src-1 ( cj293 ) * producing an activated SRC-1 molecule lacking the SH2 domain and N-terminal portion of the kinase domain, which phenotypically, results in SRC-1 overactivity.

Methods

RNA was isolated from mixed-stage animals as previously described (Tamayo et al. 2013; Paolillo et al. 2024). Poly-A selection and RNA seq library construction was conducted using the NEBnext stranded RNA seq kit. RNA seq libraries were made using the NEBNext stranded mRNA library kit. Sequencing was conducted on a Nextseq 2000 instrument with 150-bp paired-end sequencing. FASTQ files were processed using fastp (0.23.2) (Chen et al. 2018). Reads were aligned to the * C. elegans * reference genome [release WBcel235, version WBPS14 (WS271)] using HISAT2 (version 2.2.1) (Kim et al. 2015). BAM files from HISAT2 alignment were analyzed in the Integrated Genome Viewer (Robinson et al. 2011; Thorvaldsdottir et al. 2012), including Sashimi plots (Katz et al . 2010; Katz et al. 2015). Wormbase was used for * C. elegans * informatics (Sternberg et al., 2024)

Reagents

Raw FASTQ reads for * src-1 (+) * and * src-1 ( cj293 ) * were deposited in the Sequence Read Archive ( PRJNA1093133 and PRJNA1219192 , respectively). The * src-1 (+) * strain was LE5443 ( * unc-6 (lq154) X; juIs76 II * ). The balanced * src-1 ( cj293 )/+ * strain was HR1275 ( * src-1 ( cj293 ) dpy-5 ( e61 )/ hT2 I; +/ hT2 III * ).