Three SINE Insertion Polymorphic Sites were Identified in Insulin-like Growth Factor 2 mRNA-binding Proteins

Insulin-like growth factor 2 (IGF2) messenger RNA (mRNA)-binding proteins (IGF2BPs) are RNA-binding proteins (RBPs) that regulate RNA processing at multiple levels, including localization, translation and stability. IGF2BP1, IGF2BP2 and IGF2BP3 (insulin-like growth factor-2 mRNA-binding proteins 1, 2 and 3) are members of a highly conserved protein family that can bind RNA and alter the fate of their transcript targets. Many animals, including the cow (Kirby et al., 1996), sheep (Ko et al., 1991; Reynolds et al., 1997), pig (Hofig et al., 1991), horse (Lennard et al., 1995), human (Zhou et al., 1994) and rodents (DeChiara et al., 1990), have an insulin-like growth factor (IGF) system in their uterine or conceptus environment. IGFBPs are important regulators of IGF function, which influence the bioavailability and interaction of IGF-I and IGF-II with cellular receptors (Cohick and Clemmons 1993). IGF2BP1 has been reported to be the main gene responsible for the body size and plumage color of Peking ducks in a recent whole-genome analysis (Zhou et al., 2018) ÿ and novel 15 and 5 bp insertions/deletions (InDels) within the IGF2BP1 gene were identified and were found to be significantly associated with growth traits of Shaanbei white cashmere goats (Wang et al., 2020). While in sheep, nine InDels mutations within IGF2BP1 were identified, three loci were polymorphic and the three InDels were crucial variants correlated with growth traits and could be applied in marker-assisted selection (MAS) in sheep (Liu et al., 2021).
       
Transposable elements account for about 40% of the pig genome, of which retrotransposons account for more than 90% of total transposable elements (Chen et al., 2019). Retrotransposons are one of the most important sources of genetic diversity, which can produce structural changes in sequences, including deletion, inversion, displacement and breaking (Cordaux and Batzer 2009). According to comparative genomics, there are at least 8,000 structural changes associated with transposons in the human genome (Xing et al., 2009). Zhao et al., 2016 discovered that retrotransposons were responsible for more than half of the sequence deletions in the pig genome (Zhao et al., 2016). The phenotypic can be affected directly or indirectly by retrotransposons inserted into animal genes. For example, the characteristics of chicken hen feathers are related to the insertion of a complete avian leukemia virus in the 5’UTR of CYP19A1 (Li et al., 2019). The degree of dilution of the dog’s coat color will be affected by inserting the SINE retrotransposon into the exon of the PMEL gene (Murphy et al., 2018). Diverse mutations (SNPs) within MC1R, TYRP1, ASIP genes have been reported and they may be associted with phenotypes of coat color in wild pigs (Yang et al., 2019), furthermore, at least 40 RIPs in coat color genes have been reported and some RIPs may be associated with the coat color differences between different domesticated pig breeds (Du et al., 2022).
       
In summary, transposable elements are an important part of the pig genome and its insertion polymorphism is not only the raw material for molecular markers but also the entry point for studying gene structural variation. Eventhough an important functional gene of the IGF pathway, IGF2BP has no relevant research reports on whether there are structural variations mediated by retrotransposons in the IGF2BP genes in different breeds and their distribution. It is urgent to explore and reveal it to provide a basis for further research about IGF2BPs.

Experiment material
 
There were 12 pig breeds involved in this experiment, including Duroc, Landrace, Large White, Sujiang pigs, Banna pigs, Erhualian pigs, Wuzhishan pigs, Bama pigs, Tibetan pigs, Meishan pigs, Fengjing pigs and Wild boars. Ear tissues were collected in parallel to agricultural procedures (i.e., pulling in ear tags). All collected samples were stored at -80°C and detailed information is shown in Table S1. This experiment was carried out from September 2020 to August 2021 in the Laboratory of in College of Animal Science and Technology, Yangzhou University.
 

 
Primer design and synthesis
 
Primers were designed by Oligo7 software according to Fig 1A. For a SINE insertion site, the primers were designed in the flanking regions at both ends of the SINE insertion position. Only one larger band could be got for the homozygous SINE insertion (+/+) individuals and only one smaller band for homozygous SINE deletion (-/-) individuals and two bands will be got for the heterozygotes (+/+) (Fig 1B). The LINE and endogenous retrovirus (ERV) inserts involved in this article are less than 2000bp and the primers are also designed according to the SINE insertion detection method. Primers were synthesized by Nanjing Kinco Biotech Co., Ltd (Nanjing, China) and their coordinates in the reference genome, annealing temperature and sequences were supplied in Table S2. The PCR (Kaushik et al., 2017) amplification time was determined according to the target fragment size.
 

 

 
RIP annotation of porcine IGF2BP genes
 
Sequence acquisition of porcine IGF2BP genes and their flanks
 
The online website Ensembl (http://asia.ensembl.org/index.html) was used to obtain the sequences of the 3 genes of Duroc pig IGF2BP as reference sequences (IGF2BP1: ENSSSCG0000002312; IGF2BP2: ENSSSCG00000011 795; IGF2BP3: ENSSSCG00 0000366 95) and the sequence was extended to the 5'flanking region and 3'flanking region by 5000 bp and 3000 bp, respectively. NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastnandPAGE _TYPE= BlastSearchandLINK_ LOC=blasthome) was used to compare the reference sequences with the pig non-reference genomes in the WGS library and obtain the gene sequence fragments of the 3 genes of pig IGF2BP in the non-reference genomes. Finally, the sequence fragments were spliced together according to the reference sequence and a complete sequence from each genome for each gene was obtained.
 
Retrotransposon annotation
RepeatMasker (Caballero et al., 2014) (versions: 4.0.7, -cutoff 250 -nolow) was used to perform retrotransposon annotation on the gene (including flanking region) sequences of all genomes obtained and only retaining the annotations with an alignment score of more than 1000 and size more than 100 bp. Multiple sequence alignment of each gene got from 16 genomes was done by using Clustalx (2.0 version) software, then the structural variations (more than 50 bp) were counted manually. All statistics are recorded but they may not be accurate due to the uncertainty of sequencings, such as the existence of gap or long sequence N, which had not been counted. Then, the structural variations that overlap with retrotransposons over 80% of the length were identified as predicted retrotransposon insertion polymorphic sites (RIPs) and be further verified by PCR.
 
Verification for the predicted RIPs in porcine IGF2BPs gene
 
Two DNA pools were prepared for each breed after DNA was extracted from the above 12 breeds by using the Tiangen Genome Extraction Kit (DP304) kit and the extraction steps were carried out strictly following the instructions. Each pool is made up of equal amounts of DNA from three individuals and the final concentration was adjusted to 40 ng/mL. Using pool DNA as a template, PCR amplification was performed to verify each predicted RIP (Zheng et al., 2020) (Fig 2).
 

 
Conservation analysis of porcine IGF2BP genes
 
The Fasta format file of the corresponding gene sequence of each gene in 7 different species (cow, sheep, dog, horse, human, mouse. See Table S2 for detailed species information) and the annotation information of each porcine IGF2BP gene was downloaded from Ensembl. Then the conservation analysis was done using the online program of mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml). The gene sequence of the pig genome was used as a reference sequence for conservative analysis and a conservative peak map was generated in Fig S2.
 
Population genetic analysis
 
Six pig breeds information of the samples is shown in Table S1. HWE and Polymorphic information content (PIC) analysis. The genotype and allele frequencies were calculated and Hardy-Weinberg equilibrium (Jadhav et al., 2020) was tested using the chi-square test in the Popgene32 software (Yeh et al., 1999). PIC was calculated according to the formula:
         
Linkage disequilibrium for four RIPs in GHR genes was performed by Haploview (Barrett et al., 2005).

The sequence of the IGF2BPs gene and their flanks (including the 5kb 5'flanking and 3kb 3'flanking region) from the pig reference genome and 15 non-reference genomes were obtained by Blast and spliced, the genomic coordinates of the analyzed IGF2BPs genes and their flanking sequences were summarized in Table S3 and then the length of IGF2BPs genes are counted and shown in Table 1. For the IGF2BP3 gene in Göttingen pigÿit showed a long gap in the end/start after the alignment, so the effective sequence of Göttingen pig was only 55445bp. These three genes show different lengths in different genomes, indicating that there are structural variations in genes. And in Tibetan pigs, these three genes all show a relatively longer length, suggesting that they have more insertion mutations, which are different from other breeds. According to the multiple sequence alignment results obtained by Clustalx, the structural variation in each gene was counted. In IGF2BP1, IGF2BP2 and IGF2BP3 genes and their flanks, 17, 29 and 22, SVs were identified. Then, the RepeatMasker results were used for identifying the structure variation caused by retrotransposons. The results are shown in Table S3. Among these SVs, 4, 6 and 6 SVs were thought to be caused by retrotransposons which be named predicted RIPs. To confirm the mutant sequence is a retrotransposon, we cloned and sequenced the insertion allele and deletion allele sequence for the above three RIPs. As shown in Fig S1, sequencing results show that the sequence of the variation is indeed a retrotransposon which further proves that the three SVs were caused by retrotransposons. Further analysis of the retrotransposon of these RIPs, it is found that these three RIPs are all mediated by the SINEA1 retrotransposon, which was the youngest SINE in the pig genome (Chen et al., 2019) and distributed in the intron of the gene and the retrotransposon in IGF2BP3-RIP3 shows opposite direction as the gene (Fig 3) and in IGF2BP3-RIP5, IGF2BP3-RIP6 show same direction to the gene (Table 2). The RIPs are all distributed in relatively weakly conserved regions. The potential transcripts of the IG2BP3 gene sequence were predicted by Genescan and the results showed that there is a transcript in the complementary strand of the IGF2BP3 gene that is affected by whether there is a SINE in the IGF2BP3-RIP3 site. When there is a SINE presented, the third exon will be fused with a 119bp SINE sequence, resulting in the length of the third exon to be extended by 99 bp Fig S3. To further evaluate the distribution of these three RIPs in different breeds. Two commercial pig breeds (Large_White, Landrace), two native Chinese breeds (Fengjing, Erhualian) and two crossbreeds in Jiangsu province (Sujiang, Sushan) were chosen and PCR experiments were performed to detect the distribution of three RIPs in 32 individuals of each breed. The results are shown in Table 3 and Fig S4. In general, the polymorphisms of these three RIPs in commercial breeds are poor and they have been purified. Only IGF2BP3-RIP3 has a polymorphism in Landrace. The polymorphism informative contents in commercial pig breeds are lower than that in China native pig breeds, indicating that the genetic diversity of China native pigs is relatively rich, which is consistent with the analysis of SNPs and SSRs (Conson et al., 2018) and supports that molecular markers are reliable and effective for population genetic analysis.
 

 

 

 

 

 

 

 

In this study, sixteen predicted RIPs in IGF2BP genes were identified and three RIPs were verified by PCR. These three RIPs were all caused by the youngest SINEA1 retrotransposons and located in IGF2BP3 introns. The polymorphism of these RIPs in Chinese native pig breeds is higher than that in commercial pigs which is consistent with the intensive selection of commercial pigs.

This research was supported by the Jiangsu Postdoctoral Science Foundation[2021K221B] to Cai Chen, the National Natural Science Foundation of China [grant numbers 31872977 and 32002146], the Independent Innovation Fund Project of Agricultural Science and Technology in Jiangsu Province [CX (19)2016], the Priority Academic Program Development of Jiangsu Higher Education Institutions and the High-end Talent Support Program of Yangzhou University to Chengyi Song.

None.