RBFOX1

Fox-1 homolog A, also known as ataxin 2-binding protein 1 or hexaribonucleotide-binding protein 1 or RNA binding protein, fox-1 homolog, is a protein that in humans is encoded by the RBFOX1 gene.

Discovery

The RBFOX1 gene was first studied in Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio with origins in embryology and development. The derivation of the nomenclature for RBFOX1 comes from the original sexual differentiation studies in C. elegans where the gene was denoted as 'Feminizing locus On X'. This refers to a lethal splicing event which causes an increase in the chromosomal X:A ratio; feminizing XO males. In Drosophila, the gene is known as CG3206 and was noted to code for an RNA-binding protein, be affected by Notch-signaling, and be associated with non-D/V cells of the wing discs during wing development. The 'RB' portion of the gene's name extends from the RNA-binding properties of the coded protein. In zebrafish, rbfox genes were identified as being essential for cardiac and skeletal muscle development, causing reduced heart rate and paralysis respectively in morphants. The discovery of RBFOX1 in humans was due to the interaction of Rbfox1 with ataxin-2, hence the alternative name of A2BP1.

Structure

RBFOX1 is located on chromosome 16 and consists of 30 exons. The Rbfox1 protein consists of 397 amino acids and is 42,784 Da. The canonical folding of the protein includes three beta sheets and two alpha helices. The localization of Rbfox1 protein is determined by its own alternative splicing via RBFOX proteins. If exon 19 is included, Rbfox1 will be cytoplasmic, but if exon 19 is excluded, Rbfox1 will be nuclear.
There are five isoforms of Rbfox1 due to alternative splicing. The canonical variant, isoform 1, is also known as gamma. This RBFOX1 transcript includes three conserved domains in its sequence. The most clinically relevant of these domains is the RNA recognition motif located between 137-212. This domain allows for the important property of RNA binding for the Rbfox1 protein. Another conserved domain of RBFOX1 is the calcitonin gene-related peptide regulator C terminal. RBFOX1's C terminal is located between 273-363 and, as the name suggests, regulates the calcitonin gene-related peptide. The third conserved domain of RBFOX1 is the ELAV/HuD family splicing factor. HUD is human paraneoplastic encephalomyelitis antigen D whereas ELAV is Drosophila embryonic lethal abnormal visual protein. ELAV-like splicing factors are also known in humans as HuB, HuC, and HuR. This super family domain contains three RRMs and is located between 25-208.

	SNP	Alleles	AA Change	Type	Location	Associated Disease	Ref
Common	rs147023054	C>T		intron variation
Common	rs372761949	G/A	V180M	missense variation
Common	rs974157467	ACTGCCG/A		inframe deletion
Common	rs145873257	G/A	G353S	missense variation
Common	rs2093621567	CA/C		frameshift variation
Disease Relevant	rs12921846	A>T		intron variation	intron 3	Conduct Disorder in ADHD
Disease Relevant	rs10153149	A>C		intron variation	intron 3	Conduct Disorder in ADHD
Disease Relevant	rs9940753	G>C		intron variation		ADHD, ASD
Disease Relevant	rs12447542	Unknown				Schizophrenia
Disease Relevant	rs12444931	G>A		intron variation		Schizophrenia, Bipolar Disorder
Disease Relevant	rs133341055	T>G		intron variation	intron 1	Anxiety
Disease Relevant	rs809682	Unknown				Anxiety
Disease Relevant	rs142723691	A>G		intron variation		Hepatitis A
Disease Relevant	rs6500818	C>T		intron variation		Dengue Shock Syndrome
Disease Relevant	rs192187627	A>C		intron variation		COVID-19

There are forty possible isoforms but only five beyond the canonical sequence are understood and confirmed in the population. Isoform 2 of RBFOX1, also known as alpha, is a shorter form of the canonical sequence as it is missing an in-frame segment on the 3' coding region. The third variant, beta, is also a shorter version of isoform 1. This shortening is caused by an alternate exon in the 3' coding region. Because of this, isoform 3 has a differing C-terminus located between 273-360. RBFOX1's isoform 4 differs in that the 5'UTR lacks an in-frame section of the 3' coding region. This shorter isoform is encoded by variants 4 and 6 and has an alternate N-terminus. This isoform includes changes of locations of two of the conserved domains and one other domain: cell division protein ZipA becomes located between 4-122, calcitonin gene-related peptide regulator C terminal becomes located between 253-342, and the RNA recognition motif becomes located between 117-192. Isoform 5 contains a different 5'UTR as well as multiple coding region differences. Beyond these internal differences, isoform 5 also has a shorter and distinct N-terminus. The C terminus is located between 226-315 while the RRM domain is located between 117-192. The ZipA protein domain is located between 4-122. The differences of isoform 6 results in the use of an alternate start codon and a frameshift in the 3' coding region. The UTR is changed and multiple coding regions are altered. Uniquely, this isoform contains a longer rather than shorter N-terminus and a distinct C-terminus. The locations for the ZipA protein, calcitonin gene-related peptide regulator C terminal, and RRM are 33-165, 296-385, and 160-235, respectively.

Function

RBFOX1 is expressed in human heart, muscle, and neuronal tissues. The primary function is regulation of alternative splicing of associated genes. Several alternatively spliced transcript variants have been found for this gene with some localizing to the nucleus and others to the cytoplasm. Nuclear variants have a well-established role in tissue specific alternative splicing. Rbfox1 cytoplasmic variants modulate mRNA stability and translation. In stressed cells, Rbfox1 has been demonstrated to localize to cytoplasmic stress granules. Rbfox1 has an RNA recognition motif that is highly conserved among RNA-binding proteins. Rbfox1, and the related protein Rbfox2, bind the consensus RNA sequence motif GCAUG within introns to exert their functions as alternative splicing factors. The C terminus of RBFOX1 contains the code for a protein, calcitonin gene-related peptide, involved with mediating neuron-specific splicing. Together, Rbfox1 and Rbfox2 repress exon 4 inclusion. In particular, for Drosophila, two cytoplasmic Rbfox1 isoforms bind Pumilio mRNA for silencing. Because of this destabilization, germline development is promoted and reversion to earlier stages is prevented. The alternative splicing activity of RBFOX1 also aids in neuronal development specifically for CaV1.2 voltage-gated calcium channels and N-methyl-D-aspartate. The overall activity and molecular mechanism of alternative splicing mediation for RBFOX1 is not fully understood, but some qualities have been established in recent studies. For example, exon inclusion is sufficiently promoted with only the carboxy terminal tethered downstream of the alternative exon. Conversely, for repression, both the RNA binding motif and carboxy terminal are required when tethered upstream of the alternative exon. Possible proteins that aid in the inclusion or skipping process are not confirmed, though both hnRNPH and RALY have been shown to bind Rbfox1. Thus, the specific mechanisms of alternative splicing maintenance via RBFOX1 are unknown. In one study, dominant-negative RBFOX protein interfered with exon activation, though not exon skipping. Because of this knowledge, repression maintenance most likely includes other proteins or outside factors near the binding sites. In C. elegans, co-operative binding between SUP-12 and RBFOX1 is noted to account for tissue-specific splicing. In mammals, there is a more universal cooperativity between RBFOX and NOVA family of proteins. The overall repression and inclusion activity of exons via RBFOX1 seem to be positionally-related. That is, a location downstream of an intron would lead to exon inclusion and a location upstream of an intron would lead to exon exclusion.

Neurodevelopmental disorders

Autism spectrum disorder

is a neurodevelopmental disorder of social communication and repetitive behaviors as well as fixated interests and/or sensory behavior. Autism spectrum disorder is typically diagnosed in adolescence, but it is possible to be diagnosed in later stages of life. According to the DSM-5-TR, a diagnosis of Autism spectrum disorder requires at least two of the four restricted repetitive behaviors and all three verbal or nonverbal communication deficits. Mutations of RBFOX1 are not sufficient to single-handedly develop autism, but rather also require an environmental risk factor. Numerous autism spectrum disorder samples from cohorts and isolated autistic patients have been linked to de novo copy number variations of RBFOX1. Universally, cases from these studies involved intragenic deletions of either exons 5, 6, or 1D. In human progenitor cell lines modeling haploinsufficiency in neuronal differentiation, a knockdown study of RBFOX1 revealed significant changes in RNA splicing and gene expression. Similarly, whole transcriptome analysis of patients with autism spectrum disorder showcased a reduction of RBFOX1 and dysregulation of RBFOX1-dependent alternative splicing. RBFOX1 also contributes to mRNA stability of autism-related genes by blocking miRNA binding.