Accumulating evidence indicates that pre-mRNAs are co-transcriptionally spliced. However, mechanisms or factors affecting the fidelity of co-transcriptional splicing in living organisms are yet to be determined. We have previously reported use of fluorescence alternative splicing reporter worms for elucidating mechanisms of alternative splicing regulation in C. elegans. Here, we report a novel protein CCAR-1, which regulates transcription and alternative splicing. Fourteen
ccar-1 alleles were isolated in a screen for mutants defective in neuron-specific selection of exon 7a of an
unc-32 splicing reporter. With the same reporter, we have already proposed a model for the neuron-specific regulation of exon 7a by a neuron-specific CELF family splicing factor UNC-75 and RBFOX family splicing factors ASD-1 and FOX-1 (NAR, 2013). Like in the
unc-75 and
asd-1;
fox-1 mutants, exon 7b was selected instead of exon 7a in the nervous system of the
ccar-1 mutants. Unlike in the other mutants, however, double- and triple-inclusion of mutually exclusive exons
unc-32 exon 4a/4b/4c occurred in the
ccar-1 mutant. mRNAseq analysis revealed tens of alternative exons regulated by CCAR-1. Unlike in other splicing factor mutants, inclusion levels of all the affected exons were elevated in the
ccar-1 mutant except for
unc-32 exon 7a. Among such target exons was
ccar-1 exon 5, indicating that CCAR-1 negatively regulates its own expression at the pre-mRNA splicing level like many other typical splicing factors. Unexpectedly, the intron downstream from
ccar-1 exon 5 begins with evolutionarily conserved GA sequence instead of GT, and exon 5 was no longer skipped even in the wild-type worms when the GA site was changed into GT or GC in the splicing reporters. Nuclear run-on analysis revealed that co-transcriptional excision of
ccar-1 intron 4 and intron 5 was facilitated in the
ccar-1 mutant. These results indicate that CCAR-1 represses co-transcriptional splicing of exons with weak splice sites, which is critical for proper regulation of alternative splicing. mRNAseq also suggested that CCAR-1 represses abundance of its own mRNA, which was confirmed by upregulation of a
ccar-1 transcriptional fusion reporter in the
ccar-1 mutants. Chromatin immunoprecipitation (ChIP)-qPCR analyses of the wild-type and
ccar-1 mutant strains suggested that CCAR-1 is associated with its own promoter region to pause RNA polymerase II (Pol II) in the wild type. ChIP-seq analysis revealed that CCAR-1 is also associated with gene bodies of expressed genes like CTD-phosphorylated RNA Pol II, which led us to find that CCAR-1 and Pol II can be co-immunoprecipitated. These results indicate that CCAR-1 is a novel class of factors that associates with Pol II and represses transcription of its own pre-mRNA as well as co-transcriptional splicing of exons with weak splice sites to affect alternative splicing events in a variety of genes.