The spartacus phenotype was identified among G3 mice of the pedigree R6625, some of which showed reduced body weights (Figure 1) as well as reduced time on a rotarod during a rotarod performance test (i.e., impaired coordination/motor capabilities) (Figure 2) compared to wild-type littermates. Some mice also showed ataxia (Figure 3).

Whole exome HiSeq sequencing of the G1 grandsire identified 34 mutations. Both of the above anomalies were linked to mutations in three genes on chromosome 19: Scyl1, Ppp1r32, and Olfr1441. The mutation in Scyl1 was presumed causative as mutations in Scy1l are known to cause phenotypes similar to those observed in the spartacus mice (see MGI). The Scyl1 mutation is a G to T transversion at base pair 5,760,826  (v38) on chromosome 19, or base pair 10,589 in the GenBank genomic region NC_000085. The strongest association was found with a recessive model of inheritance to the body weight phenotype, wherein two variant homozygotes departed phenotypically from 26 homozygous reference mice and 41 heterozygous mice with a P value of 4.888 x 10^-11 (Figure 4).   

The mutation corresponds to residue 1,551 in the mRNA sequence NM_023912 within exon 10 of 17 total exons.

The mutated nucleotide is indicated in red. The mutation results in a valine to phenylalanine substitution at position 488 (V488F) in the SCYL1 protein, and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.999).

Figure 5. Domain organization of SCYL1. The spartacus mutation  results in a valine to phenylalanine substitution at position 488.

Scyl1 encodes Scy1-like 1 (SCYL1; alternatively, NTKL [N-terminal kinase-like] and p105), a member of the Scy1-like family of catalytically inactive protein kinases. SCYL1 has an N-terminal kinase-like domain. SCYL1 does not contain a kinase subdomain 1 (1;2). SCYL1 has three HEAT repeats and a coiled-coil. The HEAT repeats mediate SCYL1 oligomerization. SCYL1 has a C-terminal RKLD sequence that is similar to coatomer (COPI)-binding motifs in transmembrane ER proteins. The RKLD sequence mediates interactions with the appendage domain of the coatomer subunit γ-2 (alternatively, COPG2 or γ2-COP) (3).

SCYL1 undergoes alternative splicing to generate two variants that encode proteins of 791 (variant 1) and 707 (variant 2) amino acids (2). Variant 1 lacks half of exon 14, while variant 2 lacks most of exon 14, all of exon 15, and half of exon 16.

The spartacus mutation results in a valine to phenylalanine substitution at position 488 (V488F); Val488 is within an undefined region between the second and third HEAT repeats.

SCYL1 is ubiquitously expressed (1;2). SCYL1 is cytoplasmic and is localized at the interface between the Golgi apparatus and the membrane trafficking machinery mediated by coatomer (COPI)-coated vesicles (4).

The COPI complex has seven core subunits: α-COP, β’-COP, ε-COP, β-COP, δ-COP, γ-COP, and ζ-COP  (5-8). The COPI complex, along with other vesicle machinery (e.g., clathrin and COPII), promotes selection of cargo for vesicle trafficking as well as vesicle formation. The COPI complex is required for sorting of lipids and proteins within the Golgi cisternae and between the ER and Golgi (9;10). The COPI complex also functions in maintaining ER- and Golgi-resident chaperones in their respective compartments, endosomal transport and function, regulating lipid droplet homeostasis, mRNA transport, and the breakdown of the nuclear envelope [reviewed in (11)]. The COPI complex prevents the cell-surface expression of unassembled, dysfunctional proteins. The COPI complex recognizes cargo based on the presence of sorting signals within the cytoplasmic domains of the proteins (e.g., di-lysine KKxx and KxKxx motifs; arginine-based ER retrieval signals [ɸRxR; in which ɸ represents any hydrophobic amino acid]). The outer-coat COPI subunits α and β’ interact with dilysine K(X)KXX cargo motifs, and the adaptor subunit γ interacts with p23 transmembrane peptides (12-15). COPI-coated vesicles also promote the transport of K/HDEL cargoes associated with specific receptors (16); transport of HDEL-associated cargoes is dependent on δ-COP (17).

During formation of the COPI-coated vesicle, the cytoplasmic coatomer is recruited to membrane by the GTPase Arf1, which also regulate recruitment of COPI and clathrin-adaptor proteins. The F-subcomplex interacts with Arf1, subsequently recruiting the COPI complex to Golgi membranes. In the absence of cargo, inactive GDP-bound Arf1, the F-subcomplex (closed conformation), and the B-subcomplex are located in the cytoplasm. Upon cargo recognition, the B-subcomplex is recruited. Arf1 activation, via GDP-GTP exchange, promotes recruitment of the F-subcomplex via the γ-COP and β-COP subunits. Upon recruitment, the F-subcomplex transitions to an open conformation via an unknown mechanism.

SCYL1 is putatively involved in intracellular transport processes.

SCYL1 interacts with components of COPI coats (e.g., b-COP and γ-COP) to regulate COPI-mediated retrograde protein trafficking at the interface between the Golgi apparatus and the ER (3;4). RNAi-mediated SCYL1 knockdown resulted in aberrant COPI-mediated retrograde traffic of the KDEL receptor to the ER; anterograde trafficking was not affected (4). SCYL1 also interacts with class II ARFs (e.g., ARF4) to link the class II ARFs to γ2-bearing COPI subcomplexes (3).

SCYL1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery (18). SCYL1 binds tRNA and associates with the cytoplasmic side of the nuclear pore complex component Nup98 (18). SCYL1 also interacts with components of the nuclear tRNA export machinery (i.e., Xpo-t, Xpo-5, and Ran) (18). SCYL1 also putatively unloads the aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to the eukaryotic elongation factor eEF-1A for use in protein synthesis (18).

Mutations in SCYL1 are linked to cases of autosomal recessive spinocerebellar ataxia-21 (OMIM: #616719) (19;20). Patients with autosomal recessive spinocerebellar ataxia-21 show early childhood-onset of cerebellar ataxia associated with cerebellar atrophy. Patients also have recurrent liver failure episodes in the first decade of life, leading to chronic liver fibrosis and later-onset peripheral neuropathy. Some patients also show mild learning disabilities. Mutations in SCYL1 can cause recurrent early-onset low γ-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (i.e., CALFAN syndrome) (21;22).

Scyl1-deficient (Scyl1^-/-) mice showed early-onset progressive motor neuron disease with growth retardation/reduced body weights, abnormal gait (waddling), muscle wasting, reduced grip strength, and paralysis of the hind paws (23;24). Muscles from the Scyl1^-/- mice showed neurogenic atrophy, fiber type switching, and disuse atrophy (23). Scyl1^-/- peripheral nerves showed axonal degeneration and segmental demyelination (23). Scyl1^-/- mice showed a reduction in spinal ventral horn motor neuron numbers and evidence of inflammation (23). Homozygous Scyl1 mutant mice (the ‘muscle deficient’ [mdf] model) showed reduced body sizes and fertility, progressive neuromuscular atrophy, progressive reduction of forelimb grip strength, hindlimb paralysis, gait ataxia, abnormal hindlimb posture, and tremors (25-27). The mice also showed cerebellar atrophy, Purkinje cell loss, and optic nerve atrophy (27).

The phenotypes observed in the spartacus mice indicates loss of SCYL1-associated function.

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold

The following sequence of 415 nucleotides is amplified (chromosome 19, - strand):

1   ttttagacct tggttggccc tgccctggga agtggaaagt gcggtctggt ggcttggggc
61  tgcttgttgc ctctgtatat tcaagcgtcc ttttccatcc acccgggctc tcagactaga
121 cacagggtcc tcacctccgc cttcagcaga gccactaagg acccatttgc accatcccgg
181 gttgcgggtg tcctgggctt tgctgccaca cacaatctct attcgatgga cgactgtgcc
241 cataagatcc tgcctgtgct ctgtggcctt actgtggacc ctgagaaatc tgtgcgggac
301 caggtaaggc acagccaggg cagacctggg ggctgggctt ggggatattc agtctctctg
361 aggcttggcc acaccctgga agtgctgggg cctctgctac tccacaggcc tttaa

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.