CCAR-1 works together with the U2AF large subunit UAF-1 to regulate alternative splicing

Figures & data

Figure 1. CCAR-1 regulates alternative splicing. A. Methodology of RNA sequencing samples preparation between N2 and CCAR-1 null (SDW040) worms B. Types of alternative splicing events occurring from the VAST Tools analysis, including exon skipping (EX), intron retention (INT), alternative splice acceptor (ALTA) and alternative splice donor (ALTD). C. WormEnrichR analysis of gene ontology (GO) molecular function between N2 and CCAR-1 null (SDW040) worms. D. WormEnrichR analysis of gene ontology (GO) biological process between N2 and CCAR-1 null (SDW040) worms.

Table 1. Top 20 alternatively spliced genes between N2 (wildtype) and CCAR-1 null (SDW040) worms.

Caenorhabditis elegans genes	Human homolog	Alternative splicing event	Function
srap-1	MACF1	Exon Skipping	Human microtubule actin crosslinking factor 1 (MACF1), crosslinks actin to other cytoskeletal proteins and also binds microtubules.
trr-1	TRRAP	Alternative Splice Acceptor	Involved in the negative regulation of vulval development, positive regulation of growth rate and reproduction.
unc-82	NUAK1	Exon Skipping	Serine threonine kinase important for maintaining myosin filament organization.
rskn-1	RPS6KA1	Alternative Splice Acceptor	Ribosomal protein S6 kinase involved in signal transduction and protein phosphorylation.
F13H8.5	N/A	Exon Skipping	Enriched in ADE sheath, germline precursor cell and hypodermis.
sax-7	L1CAM	Intron Retention	Conserved transmembrane cell adhesion molecule involved in development of distict neurone, dendrite development, and axon guidance.
lea-1	KIAA1881	Exon Skipping	Protects cells against various abiotic stresses such as high salinity, desiccation and heat and cold stress.
F30F8.10	NAA60	Intron Retention	Enables histone acetyltransferase activity and peptide alpha-N-acetyltransferase activity.
T24B8.7	ENSP00000385700	Alternative Splice Acceptor	Enables cysteine-type deubiquitinase activity and cysteine-type endopeptidase activity.
apr-1	APC	Alternative Splice Acceptor	APC-related protein involved in embryogenesis and vulval development, regulation of Wnt signalling pathway, and regulation of multicellular organismal development.
alp-1	APLP1	Exon Skipping	APP family member of proteins that facilitates heparin binding activity and transition metal ion binding activity. Involved in body morphogenesis, ecdysis, collagen and cuticulin-based cuticle, and nematde larval development.
unc-70	HSpTB1	Exon Skipping	Facilitates actin filament binding activity and is involved in dendrite development, embronic body morphogenesis, and muscle cell celular homoeostasis.
K04H4.2	N/A	Exon Skipping	Enables chitin binding activity.
unc-103	ENSP00000396900	Exon Skipping	Enables inward rectifier potassium channel activity. Involved in mating behaviour and regulation of muscle contraction.
zyg-12	HOOK1	Exon Skipping	Involved in centrosome localization, embryo development, and pronuclear migration.
mlt-11	TFPI	Exon Skipping	Serine-type endopeptidase inhibitor activity expressed in hypodermis.
daf-15	RPTOR	Alternative Splice Acceptor	Protein kinase activator and protein-macromolecule adaptor involved in dauer larval development, determination of adult lifespan, and regulation of autophagosome assembly. Expressed in the body wall musculature and pharyngeal muscle cell.
pat-12	N/A	Exon Skipping	Involved in embryonic morphogenesis and hemidesmosome assembly. Located in hemidesmosome.
ect-2	ECT2	Alternative Splice Acceptor	Epithelial cell transformation, cell fate determination, mitotic cytokinesis, and positive regulation of GTPase activity.
tim-1	TIMELESS	Alternative Splice Acceptor	DNA binding activity, embryo development, and sister chromatid cohesion.

Figure 2. RT-PCR validation of alternatively spliced genes. This figure shows the RT391 PCR validation of CCAR-1-regulated alternative splicing from the RNA sequencing dataset. A. The RT-PCR images show the visual representation of isoform changes in the N2 and CCAR-1 null (SDW040) worms. B. The box plots represent the distribution of the isoform ratios of all populations of N2 and CCAR-1 null (SDW040) worms, N=7. The F30F8.10 box plot measures the ratio of isoform A to C and the tos-1 box plot measures the ratio of isoform A to B. Based on a Mann-Whitney test, we recorded a p-value of 0.006 (***) for the F30F8.10 gene and a p-value 0f 0.0401 (*) for the tos-1 gene.

Figure 3. CCAR-1RNAi affects the alternative splicing of the tos-1 splicing reporter in UAF-1 mutants. A. Shows the tos-1 pre-mRNA and subsequent isoforms due to alternative splicing B. The RT-PCR results of N2, CCAR-1 null (SDW040), UAF-2 mutant (VC3010), and UAF-1 mutant (MT16492) (with and without ccar-1 RNAi). C. Shows the isoform ratios of isoform a to D in N2, CCAR-1 null (SDW040), UAF-2 mutant (VC3010), and UAF-1 mutant (MT16492) (with and without ccar-1 RNAi) D. Shows the isoform ratios of isoform C to D in N2, CCAR-1 null (SDW040), UAF-2 mutant (VC3010), and UAF-1 mutant (MT16492) (with and without ccar-1 RNAi). The error bars in C and D represent the standard error and variability of isoform ratios from different replicates.

Figure 4. CCAR-1RNAi rescues the motility of UAF-1 mutants. N2 and UAF-1 mutant (MT16492) grown at 25°C, were assessed for motility in a thrashing assay. Body bends per minute were measured for individual worms in an N = 15 for two biological replicates. Based on a Mann-Whitney test, we recorded a p-value of < 0.0057 between N2 EV and N2 ccar-1 RNAi and a p-value of < 0.0001 between UAF-1 mutants (MT16492) EV and UAF-1 mutants (MT16492) ccar-1 RNAi.

Figure 5. Ccar-1 and uaf-1 RNAi significantly decrease polyglutamine aggregation. A. Shows the fluorescent images of AM140 (Q35:YFP) fed control, ccar-1, uaf-1, and ccar-1/uaf-1 RNAi from L1 larval stage to day 3 of adulthood. B. Shows quantification of the number of aggregates per worm in each condition with an N = 40. Based on a Mann-Whitney test, we recorded a p-value of 0.0001 (***) between AM140 empty vector (EV) and AM140 ccar-1 RNAi, and a p-value of 0.0001 (***) between AM140 uaf-1, and AM140 uaf-1/ccar-1 RNAi.

Figure 6. A model of the mechanism of action of alternative splicing regulation by CCAR-1 and UAF-1 inC. elegans.

Data availability statement

The data that supports the findings of this study are openly available in the Sequence Read Archive (SRA) website https://submit.ncbi.nlm.nih.gov/subs/sra, reference number SUB8460771.