Comparison of RNF180 amino acid sequences in different species.
Ten evolutionary representative organisms from lower vertebrates to higher mammals were used for RNF180 amino acid sequence comparisons. These species included Zebrafish, Xenopus tropicalis, Chicken, Zebra finch, Cow, Rat, Mouse, Orangutan, Chimpanzee and Human. Our results showed that conserved RNF180 gene sequences gradually increased from lower vertebrates to primates, and the RNF180 sequence similarity between Humans and Chimpanzees is 99.0% (Table.2). With the exception of Cow, the species RNF180 sequence alignment results revealed an evolutionary relationship consistent with that described by the evolutionary tree (Fig. 2). The RNF180 C-terminal amino sequence was highly conserved as were those of exons 3, 6, and 7. Exon 1 and exon 8 were not translated most of the examined species. Exon 2 (1–45) was missing in Zebra finch and us partially deleted in Chicken and X. tropicalis. Exon 4 (78–398) was not well conserved in Zebrafish and Chicken, X. tropicalis, and Zebrafish were devoid of exon 5 (399–411) (Supplementary Fig. S1). Further analysis using the COBALT online multiple sequence alignment analyzer revealed considerable variability at amino acid sites 151, 175, 221, 254, 361, 400, and 531.
Table 2
Human RNF180 sequence similarity comparison with other species.
Species
|
Length
|
Species
|
ID
|
Length
|
Score(%)
|
Human
|
592
|
chimpanzee
|
ENSPTRP00000028966
|
592
|
99.0
|
Human
|
592
|
orangutan
|
NP_001125710.1
|
592
|
98.0
|
Human
|
592
|
cow
|
NP_001192746.1
|
592
|
87.0
|
Human
|
592
|
rat
|
NP_001128458.1
|
592
|
83.0
|
Human
|
592
|
mouse
|
NP_082210.1
|
575
|
80.0
|
Human
|
592
|
chicken
|
ENSGALP00000023728
|
571
|
51.0
|
Human
|
592
|
zebra finch
|
ENSTGUP00000002858
|
532
|
51.0
|
Human
|
592
|
x.tropicalis
|
NP_001107726.2
|
558
|
43.0
|
Human
|
592
|
zebrafish
|
NP_001103874.1
|
458
|
34.0
|
Evolutionary selection pressure (dN/dS) analysis.
dN/dS analysis was performed on RNF180 exons 2–8 (Exon 1 is only present in individual higher mammals, and the sample size was insufficient for comparison). The results showed that the dN/dS values of exons 7 and 8 are > 1 (1.49825 and 1.23845), suggesting positive selection. Exons 1–6 with dN/dS < 1 were stably selected, and exon 6 was selected for purification (dN/dS = 0.160536 < 0.25) (Fig. 3 and Table.3).
Table 3
dN / dS analysis of eight exons of the RNF180 gene.
Exon
|
dN / dS
|
Exon2
|
0.334887
|
Exon3
|
0.38672
|
Exon4
|
0.372754
|
Exon5
|
0.447605
|
Exon6
|
0.160536
|
Exon7
|
1.49825
|
Exon8
|
1.23845
|
Analysis of alternative splicing of the RNF180 gene in different species.
We downloaded the standard reference RNF180 genes from a range of species from lower vertebrates to higher mammals using the NCBI database and the Ensembl Gene Browser. From these data, we predicted AS isoforms in Human, Chimpanzee, Mouse, Rat, Cow, Chicken, X. tropicalis and Zebra finch. The Orangutan, Guinea pig, and Zebrafish sequences did not match our query transcripts (Table.4). The Human RNF180 genome annotation was provided by the UCSC Genome Browser. The longest transcript, transcript 1, had eight exons. For transcript 2, the TGA premature termination codon was introduced in exon 5. As for transcript 3, a jump of exon 4 to exon 3 was observed and a premature TGA termination codon was introduced in exon 5. A premature TAA termination codon was also observed in exon 7 of transcript 4. The CDS sequences of human RNF180 gene were used as a reference. Comparison with RNF180 sequences on other species revealed that the main splicing patterns in these species are consistent with that of human. Exons 7 and 8 terminate prematurely, exon 4 jumping only was observed in human, and exon 5 was missing in multiple species (Supplementary Fig. S2 and Table.5). Additionally, RNF180 splicing in Chicken, X. tropicalis and Zebra finch was complicated and requires further study.
Table 4
RNF180 gene alternative splice in species.
Species
|
Transcript number
|
Total
|
Database
|
human
|
NM_001113561.2; NM_178532.4; NM_001323291.1; NM_001323292.1; XM_017009383.1; XM_017009384.1;
XM_017009385.1; XM_017009386.1; XM_017009387.1; XM_017009388.1; XM_017009389.1
|
11
|
NCBI
|
chimpanzee
|
XM_009449113.2; XM_527201.6; XM_009449114.2
|
3
|
NCBI
|
mouse
|
NM_027934.2; XM_006517761.3; XM_011244696.2; XM_006517756.3; XM_006517757.2; XM_006517759.3;
XM_006517760.2; XM_006517762.2; XM_017315607.1
|
9
|
NCBI
|
Rat
|
NM_001134986.1; XM_008760714.1
|
2
|
NCBI
|
cow
|
NM_001205817.1; XM_010816737.2
|
2
|
NCBI
|
chicken
|
XM_015277600.1; XM_015277601.1; XM_004937275.2; XM_004937273.2; XM_015277602.1
|
5
|
NCBI
|
X. tropicalis
|
NM_001114254.2; XM_012954387.2; ENSXETT00000032412.3
|
3
|
NCBI Ensembl
|
Zebra finch
|
XM_012577664.1;
ENSTGUT00000002887; ENSTGUT00000002889
|
3
|
NCBI
Ensembl
|
Table 5
Splicing methods of different species.
Species
|
Transcript number
|
Alternative method
|
human
|
XM_017009384.1; XM_017009385.1; NM_001323291.1
NM_178532.4; NM_001323291.1; XM_017009387.1;
NM_001323292.1; XM_017009386.1
|
exon2 skip; exon4 skip;
exon5 introduces the forward-termination codon TGA;
alternative poly-A site;
exon7 introduces the forward-termination codon TAA
|
chimpanzee
|
XM_009449114.2; XM_009449113.2
|
alternative promoter; exon2 skip
|
mouse
|
XM_006517762.2; XM_006517761.3; XM_006517757.2; XM_017315607.1; XM_006517759.3; XM_006517756.3
XM_006517760.2; XM_011244696.2; NM_027934.2
|
alternative promote;
exon8introduces the forward-termination codon TGA
|
Rat
|
XM_008760714.2
|
exon3 skip
|
cow
|
XM_010816737.2; NM_001205817.1; XM_010816737.2
|
exon2 skip; exon5 skip
|
RNF180 intron sequence comparison in multiple species.
The UCSC Genome Browser provides human RNF180 genome annotation and reference sequences for other species, including Cow, Chimpanzee, Mouse, and Rat. Cow, Chimpanzee, Mouse, and Rat lack exon 5, while Cow, Mouse, Rat also lack exon 1, and exon 8 is missing in Mouse. These results were consistent with those of previous RNF180 gene sequence comparisons (Figs. 4 and 5). With reference to the Human RNF180 intron sequences, we analyzed RNF180 from multiple species (Chimpanzee, Cat, Dog, Chicken, X. tropicalis, Zebrafish, Zebrafish, and other species) using Clustalx. We found that Human and Gorilla had the most conserved intron sequences, and that exon 5 was absent in Chimpanzee, Cow, Cat, Dog, Chicken, X. tropicalis, Zebrafish, Armadillos, Elephant, and Guinea pig. Therefore, we sought to determine the evolutionary origin of the human exon 5 sequence by comparing it with the corresponding intron region sequences in other species. Our results showed that the conservation between Human and Gorilla, Cow, Cat, Armadillo, Elephant, and Dog were 100%, 96%, 94%, 91%, 88%, and 86%, respectively (Table.6). In silico analysis revealed that most of the frequent mutations were predicted to be deleterious (Supplementary Table S1).
Table 6
Contrast between Exon5 and corresponding Intron.
Name
|
Length
|
Name
|
Length
|
Score(%)
|
exon5
|
36
|
Gorilla
|
112248
|
100
|
exon5
|
36
|
Cow
|
135704
|
96
|
exon5
|
36
|
Cat
|
87272
|
94
|
exon5
|
36
|
Armadillo
|
147264
|
91
|
exon5
|
36
|
Elephant
|
149778
|
88
|
exon5
|
36
|
Dog
|
87296
|
86
|