Phenotypic Mutation 'curlyfry' (pdf version)
Allelecurlyfry
Mutation Type critical splice acceptor site
Chromosome1
Coordinate160,786,969 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Rc3h1
Gene Name RING CCCH (C3H) domains 1
Synonym(s) roquin, 5730557L09Rik
Chromosomal Location 160,733,988-160,802,548 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a protein containing RING-type and C3H1-type zinc finger motifs. The encoded protein recognizes and binds to a constitutive decay element (CDE) in the 3' UTR of mRNAs, leading to mRNA deadenylation and degradation. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2014]
PHENOTYPE: A single recessive mutation on this gene resulted in severe autoimmune disease with phenotype resembling human systemic lupus erythematosus. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_027902; MGI:1919003

MappedYes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000037178 ] [ENSMUSP00000124871 ]   † probably from a misspliced transcript
AlphaFold Q4VGL6
SMART Domains Protein: ENSMUSP00000037178
Gene: ENSMUSG00000040423

DomainStartEndE-ValueType
RING 14 53 5.9e-8 SMART
low complexity region 201 212 N/A INTRINSIC
Pfam:zf-CCCH 414 440 1.4e-4 PFAM
low complexity region 551 562 N/A INTRINSIC
low complexity region 626 636 N/A INTRINSIC
low complexity region 728 750 N/A INTRINSIC
low complexity region 770 784 N/A INTRINSIC
coiled coil region 954 983 N/A INTRINSIC
low complexity region 994 1002 N/A INTRINSIC
low complexity region 1084 1098 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000037178
Gene: ENSMUSG00000040423

DomainStartEndE-ValueType
RING 14 53 5.9e-8 SMART
low complexity region 201 212 N/A INTRINSIC
Pfam:zf-CCCH 414 440 1.4e-4 PFAM
low complexity region 551 562 N/A INTRINSIC
low complexity region 626 636 N/A INTRINSIC
low complexity region 728 750 N/A INTRINSIC
low complexity region 770 784 N/A INTRINSIC
coiled coil region 954 983 N/A INTRINSIC
low complexity region 994 1002 N/A INTRINSIC
low complexity region 1084 1098 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000124871
Gene: ENSMUSG00000040423

DomainStartEndE-ValueType
RING 14 53 1.25e-5 SMART
low complexity region 201 212 N/A INTRINSIC
Pfam:zf-CCCH 414 440 5.3e-7 PFAM
low complexity region 551 562 N/A INTRINSIC
low complexity region 626 636 N/A INTRINSIC
low complexity region 728 750 N/A INTRINSIC
low complexity region 770 784 N/A INTRINSIC
coiled coil region 954 983 N/A INTRINSIC
low complexity region 1003 1011 N/A INTRINSIC
low complexity region 1093 1107 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000124871
Gene: ENSMUSG00000040423

DomainStartEndE-ValueType
RING 14 53 1.25e-5 SMART
low complexity region 201 212 N/A INTRINSIC
Pfam:zf-CCCH 414 440 5.3e-7 PFAM
low complexity region 551 562 N/A INTRINSIC
low complexity region 626 636 N/A INTRINSIC
low complexity region 728 750 N/A INTRINSIC
low complexity region 770 784 N/A INTRINSIC
coiled coil region 954 983 N/A INTRINSIC
low complexity region 1003 1011 N/A INTRINSIC
low complexity region 1093 1107 N/A INTRINSIC
Predicted Effect probably null
Meta Mutation Damage Score 0.9499 question?
Is this an essential gene? Possibly nonessential (E-score: 0.298) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(12) : Chemically induced (ENU)(3) Gene trapped(2) Radiation induced(1) Targeted(6)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
sanroque APN 1 160940830 synonymous probably benign
IGL00417:Rc3h1 APN 1 160783551 critical splice donor site probably null
IGL02302:Rc3h1 APN 1 160765675 splice site probably benign
IGL03053:Rc3h1 APN 1 160783387 missense probably benign
IGL03275:Rc3h1 APN 1 160787125 critical splice donor site probably null
PIT4651001:Rc3h1 UTSW 1 160791110 missense probably benign 0.04
R0528:Rc3h1 UTSW 1 160795228 missense probably damaging 1.00
R0609:Rc3h1 UTSW 1 160757705 missense probably damaging 1.00
R1620:Rc3h1 UTSW 1 160782543 missense probably benign 0.02
R1661:Rc3h1 UTSW 1 160786993 missense probably benign 0.29
R1665:Rc3h1 UTSW 1 160786993 missense probably benign 0.29
R2027:Rc3h1 UTSW 1 160782507 missense probably benign 0.03
R2145:Rc3h1 UTSW 1 160757827 missense probably damaging 1.00
R2207:Rc3h1 UTSW 1 160767595 missense probably damaging 0.97
R2227:Rc3h1 UTSW 1 160791112 missense probably benign 0.07
R2348:Rc3h1 UTSW 1 160778430 missense probably damaging 1.00
R2925:Rc3h1 UTSW 1 160782546 missense probably damaging 1.00
R3977:Rc3h1 UTSW 1 160786969 critical splice acceptor site probably null
R5071:Rc3h1 UTSW 1 160787047 missense possibly damaging 0.76
R5177:Rc3h1 UTSW 1 160779222 missense probably damaging 1.00
R5410:Rc3h1 UTSW 1 160792533 missense possibly damaging 0.47
R5421:Rc3h1 UTSW 1 160779400 critical splice donor site probably null
R5699:Rc3h1 UTSW 1 160757823 missense probably damaging 1.00
R5873:Rc3h1 UTSW 1 160787071 missense probably damaging 0.99
R7672:Rc3h1 UTSW 1 160778454 missense probably damaging 0.99
R8163:Rc3h1 UTSW 1 160782629 missense probably damaging 1.00
R8271:Rc3h1 UTSW 1 160768329 intron probably benign
R8424:Rc3h1 UTSW 1 160793342 missense probably damaging 1.00
R8746:Rc3h1 UTSW 1 160757744 missense probably damaging 1.00
R8805:Rc3h1 UTSW 1 160795222 missense probably benign 0.10
R8960:Rc3h1 UTSW 1 160774164 missense probably damaging 0.98
R8980:Rc3h1 UTSW 1 160782595 missense probably benign 0.11
R9011:Rc3h1 UTSW 1 160792673 missense probably damaging 1.00
R9688:Rc3h1 UTSW 1 160770234 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice
Repository
Last Updated 2019-09-04 9:44 PM by Anne Murray
Record Created 2015-10-13 2:36 PM by Danielle White
Record Posted 2016-01-20
Phenotypic Description
Figure 1. The curlyfry phenotype.
Figure 2. Curlyfry mice exhibit reduced body weights compared to wild-type (REF) mice. Scaled body weights are shown. Abbreviations: REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The curlyfry phenotype was identified among G3 mice of the pedigree R3977, some of which showed curly tails (Figure 1) and reduced body weights compared to their wild type littermates (Figure 2).

Nature of Mutation

Figure 3. Linkage mapping of the body weight phenotype using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 52 mutations (X-axis) identified in the G1 male of pedigree R3977. Weight phenotype data are shown for single locus linkage analysis with consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 52 mutations. The tail and body weight phenotype were linked to two genes on chromosome 1: Tnr and Rc3h1. The mutation in Rc3h1 is presumed to be causative because the curly tail and body weight phenotype mimics other known alleles of Rc3h1 [see MGI for a list of Rc3h1 alleles and (1)]. The mutation in Rc3h1 is an A to G transition at base pair 160,959,399 (v38) on chromosome 1, or base pair 52,989 in the GenBank genomic region NC_000067 encoding the Rc3h1 gene. The strongest association was found with a recessive model of linkage to the body weight phenotype, wherein seven variant homozygotes departed phenotypically from 21 homozygous reference mice and 25 heterozygous mice with a P value of 4.935 x 10-10 (Figure 3). A substantial semidominant effect was observed in the body weight assay, but the mutation is preponderantly recessive. The mutation is located within the acceptor splice site of intron 13, two nucleotides from exon 14. The effect of the mutation at the mRNA and protein level is unknown. One possibility, is that a cryptic site in intron 13 may be used, resulting in a 28-base pair insertion of intron 13. The insertion would cause a frame-shift (affecting the protein after amino acid 788) and subsequent premature termination after the inclusion of 12 aberrant amino acids. 

             <--exon 13         <--intron 13 exon 14-->               <--exon 20
49555 ……TTCCATCCAGAGGAA ……tattttccccctctatag TTTTTGGATGAAGAC…… ……AATTCTGCTCCCTAG…… 61387
784   ……-F--H--P--E--E-                      -F--L--D--E--D-…… ……-N--S--A--P--*-   1130
                                         correct

             <--exon 13                   <--intron 13 exon 14-->                 

49555 ……TTCCATCCAGAGGAA ……acccatttcctattttccccctctatgg TTTTTGGATGA…… 53001
784   ……-F--H--P--E--E- ……-T--H--F--L--F--S--P--S--M-- V--F--G--*-   800                 
           correct                      aberrant
 
Genomic numbering corresponds to NC_000067. The acceptor splice site of intron 13, which is destroyed by the mutation, is indicated in blue; the mutated nucleotide is indicated in red.
Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 4. Roquin-1 functional domains. The location of the curlyfry mutation is indicated. Abbreviations: CC, coiled-coil; RING1, E3 ubiquitin ligase zinc finger domain; ROQ, novel ROQ domain; CCCH, RNA-binding zinc finger.

Rc3h1 encodes the highly conserved 1,130-amino acid intracellular Roquin-1 protein. The protein contains several domains (Figure 4), including an N-terminal RING-1 zinc finger at residues 14-53, a highly conserved novel protein domain (ROQ domain) at amino acids 131-360, a CCCH-type zinc finger domain at residues 419-438, a proline-rich region at amino acids 533-786, and a coiled-coil domain at amino acids 954-981 (1).

The curlyfry mutation affects the acceptor splice site of intron 13 and is predicted to lead to inclusion of 12 aberrant amino acids followed by a premature stop codon after amino acid 800. Amino acid 800 is C-terminal to the proline-rich region.

For more information about Rc3h1, please see the record for sanroque.

Putative Mechanism

Roquin-1 has a RING finger zinc domain typical of the E3 ubiquitin ligase family. RING finger proteins covalently attach ubiquitin to a lysine on target proteins via an isopeptide bond (2). It is possible that Roquin-1 may function as an E3 ubiquitin ligase, but its targets remain unknown. Roquin-1 is critical for negatively regulating ICOS expression and appears to do so post-transcriptionally by mediating Icos mRNA degradation (3). B cells interact with TFH through the pairing of T cell and B cell surface ligands and receptors such as CD40 with its ligand, CD28 with B7 ligands, and ICOS with ICOS ligand (ICOSL). This interaction results in the secretion by TFH cells of cytokines particularly IL-10 and IL-21, which are known to promote B cell survival, proliferation, and antibody production (4;5). IL-21 is also critical for the formation of TFH cells after immunization (3;6)

Rc3h1-deficient (Rc3h1-/-) mice exhibit perinatal lethality as well as a curly tail and malformations of the caudal spinal column, indicative of delayed or abnormal neural tube closure (7). The perinatal lethality was proposed to be due to impaired lung function (7). Rc3h1-/- mice exhibit reduced postnatal body weights from 14 to 55 weeks of age (8). The curlyfry mice exhibit curly tails and reduced body weights similar to Rc3h1-/- mice, indicating loss of Roquin-1 function. Other immune-related phenotypes observed in the sanroque mice (1) (e.g., the production of antinuclear autoantibodies, focal proliferative glomerulonephritis, necrotizing hepatitis, anemia, autoimmune thrombocytopenia, lymphadenopathy, and splenomegaly) have not been examined in the curlyfry mice.

Primers PCR Primer
curlyfry_pcr_F: TTGCTGAAGGAAGTTGAAACTC
curlyfry_pcr_R: CAAAAGCAAAGCTCAATTAGTTGTCCC

Sequencing Primer
curlyfry_seq_F: AGCTGCCATTAACAAATATGAAAGC
curlyfry_seq_R: GTCCTGGAACTCACTTGTAGACCAG
Genotyping

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 717 nucleotides is amplified (chromosome 1, + strand):


1   ttgctgaagg aagttgaaac tcaatcaatc aatatctctc tctctctctc tctctctctc
61  tctctctctc tctctctccc cctccatctc cctccatcca tcccttcctc cttccctccc
121 ttccttcctt cctttttttt tagctgccat taacaaatat gaaagctaaa ataatcttca
181 catttttcag tctataaaaa tatgttctga aacttaataa tcaaataact atcactatat
241 tgttttaagt tggtattcta gacattccac gctttttctg ctttgttaac tcatagaccc
301 atttcctatt ttccccctct atagtttttg gatgaagact tgaaggtagc cggaaaatac
361 aaagcaaatg attatagcca gtattctcct tggtcatgtg ataccatcgg ctcctacatt
421 ggaaccaaag atgcaaaacc caaagatgtt gtggcagcgg ggagtgtgga aatgatggta
481 tgtgtaatgg ctaaacataa ttaatagaaa tctagataca gccgggcgtg gtggcgcatg
541 cctttaatcc cagcacttgg gaggcagaag caggtggatt tctgagttcg aggccagcct
601 ggtctacaag tgagttccag gacagccagg gctatacaga gaaaccctgt ctcgaaaaaa
661 aaaaataaag aagaagaaaa aaaaattata gggacaacta attgagcttt gcttttg


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

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsDanielle White, Lauren Prince, Jamie Russell, Emre Turer, and Bruce Beutler