| RNA editing is a process of post-transcriptional modification of RNA,including insertion,deletion,and replacement of bases.By far the most common RNA editing is base substitution,and the most common RNA editing is A-to-G catalyzed by adenylate deaminase(ADAR)and C-to-T catalyzed by the RNA editing enzyme apo lipoprotein B(APOBEC).The direct result of RNA editing is the change of base,and the editing of RNA in different sites(coding and non-coding regions)usually has different effects.Nonsynonymous editing events that occur in a coding region usually result in changes in protein function that introduce new protein isomers to increase proteome diversity.Cave fish live in cave or underground rivers for a long time.They gradually develop adaptive characteristics to adapt to the cave environment with no light,low oxygen and lack of food.In this study,two typical cavefish species(Triplophysa rosa and Astyanax mexicanus)were used as research objects.T.rosa(Teleostei:Cypriniformes: Nemacheilidae)is a typical cave fish,which lives in underground caves for a long time.It has linear cavefish characteristics,highly deformed eyes,albino and transparent whole body.Some T.rosa did not completely lose the ability to synthesize melanin,which is called melanin-maintain type.The other T.rosa kept albino body color no matter how long it was cultured under natural light,which is called albino type.A.mexicanus(Teleostei: Characiformes: Characidae)is mainly composed of two kinds of phenotype,the first ancestors of a class type,with normal eyes,mainly live in the rivers and streams,the second type is derived from the surface mprhps,showed the lack of eyes and body color bleaching,mainly live in the cave northeast of Mexico.In this study,the distribution of RNA editing events in different phenotypes and tissues of A.mexcanius and T.rosa,to analyze whether there is a direct relationship between RNA editing and body color degradation of T.rosa and eye disappearance of A.mexcanius.This study aims to explore the adaptation of cavefish to the environment from the perspective of RNA editing,and provides new clues to uncover the mechanism of cavefish environmental adaptation.We also analyzed ADARs family in fish to get insight into the distributions of ADARs and pave the way to unveil the function of RNA editing and RNA editing enzymes in fish.By identifying RNA editing events from transcriptomes of T.rosa and A.mexcanius,we found something significant.For T.rosa,there was no significant difference in the number of RNA editing events in the brain and skin samples of the melanin-retention type and the albino type(Skin of melanin morph:MS＿T02: 12027,MS＿T04: 12052;skin of albino morph: AS＿T08: 11074,AS＿T09: 12902;brain of melanin morph: MB＿T01: 11232,MB＿T03: 10799;brain of melanin morph: AB＿T07:11179,AB＿T10: 11862),but the number of edit clusters in the brain was significantly more(brain: 513,skin: 110).There was a significant difference in the number of RNA editing events between the geophenotype of A.mexcanius and the eyes of the cave type.The number of RNA editing events in the cave morhps was almost 6 times that of the surface morhps(Cave＿1: 25223,Cave＿2: 34601,Surface＿1: 4694,Surface＿2: 8449).Most of the RNA editing events occurred in the non-coding regions(the intergenic region,introns,downstream of the gene,gene upstream,3’URT,5’UTR),only a small part of RNA editing is a heavy coding function,which is located in the coding regions(such as the editing sites of exon area,causing the wrong mutations,termination codon,termination codon loss,etc.).From an evolutionary point of view,the RNA editing sites were positively selected(d N/d S >1)in both species,but the positive selection was stronger in the T.rosa(d N/d S mean = 2.38)than in the A.mexcanius(d N/d S mean = 1.08).Moreover,d N/d S in the skin of the T.rosa was significantly higher than that in the skin(P < 0.01),which indicated that RNA editing events suffered stranger selective pressure in skin.In order to understand the specific function of the edited genes,GO enrichment analysis was performed on the edited genes in A.mexcanius and T.rosa.In GO analysis of the edited genes in the eyes of cave A.Mexcanius,it was found that a large proportion of the edited genes were enriched in methylation related items,but these items were not enriched in surface morphs.The genes enriched in methylation were not differentially expressed between cave and surface morphs.The main difference was that the genes were edited in only cave type rather than in surface morphs.In terms of GO enrichment of enriched genes in T.rosa,we found that pigment-related items were enriched in the brain and skin tissues of melanin-maintain T.tosa ranther than in albino type.There was no difference in the expression of these pigment-related genes between the two types of T.rosa,and the main difference was that these pigment-related genes were only edited in the melanin-retaining type of T.rosa.A total of 28 edited conserved genes were identified in T.rosa and A.mexcanius.When analyzing the functional structure of the proteins corresponding to these genes,we found that the editing events on the functional domain are few indeed,and most of them are synonymous editing.We focused on editing sites within coding regions,and found that there are more RNA editing events in the disordered protein region.This means that RNA editing may not change the protein’s sequence,but it may affect the folding state of the protein.RNA editing tends to occur on repeats,and transposons,as a special class of repeats,are widely distributed in fish.To investigate whether RNA editing tends to occur in transposons,the present study identified editing events occurring in transposons.We found that there are cases where multiple transposons share an RNA edit and where multiple RNA editing events are located on the same transposon.Most RNA editing events are shared by multiple transposons in T.rosa,while it is common for multiple RNA editing sites to be located on the same transposon in A.mexcanius.In order to have a deeper understanding of the RNA editing enzyme ADARS family in fish,48 species of fish were selected for analysis,and Branchiostoma belcheri was selected as the outgroup.There are four members of the ADARs family in fish(adar,adarb1 a,adarb1b,and adarb2).Through phylogenetic analysis,we found that the members of the adars family clustered into three major groups,namely,adar,adarb1(including adarb1 a,adarb1b)and adarb2.Among them,the largest number of adarb1,and adar and adarb2 accounted for a smaller proportion.Only 20 species of bony fish have been found to have adarb1 a.The first species to evolve adarb1 a was Gonorynchiformes(Chanos Chanos).Adarb1 a has not been identified in older species such as Erpetoichthys calabaricus and Lepisosteus oculatus.To investigate the effect of chromosomal multiplier changes on the copies of members of the Adars family,we analyzed the copy number of family members in diploid and tetraploid fish.We found that chromosome doubling directly affected the copies of adar and adarb1 a members in the ADARs family,but had less effect on adarb1 b and adarb2.And for the diploid species with only single copy of adar,adarb1 a,adarb1b and adarb2,the genes near adar were highly conserved,but the conserved degree was low in tetraploid species.In conclusion,our study analyzed the RNA editing events of A.mexcanuis and T.rosa and found that RNA editing might indirectly participate in cave adaptation by affecting some important genes or gene sets(methylation-related gene sets in A.mexcanus and pigment-related gene sets in T.rosa)in cave fish.This study found that RNA editing may affect pigment-related pathways and thus participate in the body color degradation process of T.rosa,The presence of more RNA editing events in the eyes of the cave type of A.mexcanus as well as the occurrence of RNA editing in a large number of methylation related genes,suggest that RNA editing may be involved in the degeneration of the eyes of A.mexcanus.The analysis of distribution and copy of ADAR family members in fish provides a basic insight for our further analysis of RNA editing events and their editing enzymes in different kinds of fish. |
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