Intramuscular fat deposition in the longissimus dorsi
muscle (LM) of Korean cattle steer is regulated by several genes related to
lipid metabolism. One of these genes encodes the enzyme bovine
glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), which is located on
the mitochondrial outer membrane and catalyzes the initial and committed
step of glycerolipid synthesis in lipid metabolism of cattle. Previous
studies have shown that the 3′-untranslated region (UTR) of the
GPAM is quite extended and contains a polyadenylation signal site, erythroid
15-lipoxygenase differentiation control elements (15-LOX-DICEs), and
cytoplasmic polyadenylation elements (CPEs) that affect the regulation of
triacylglycerol synthesis. Therefore, the aim of this study was to
identify single-nucleotide polymorphisms (SNPs) related to the regulation of
glycerolipid synthesis in the 3′-UTR of GPAM and to verify the function
of SNPs affecting the deposition of intramuscular fat in Korean cattle
steer. In the present study, 11 SNPs were discovered in the 3′-UTR
of GPAM. Among these SNPs, g.54853A>G, g.55441A>G, and
g.55930C>T were significantly associated with marbling score in
a Korean cattle steer population and were strongly correlated with each other
within the GPAM gene. Furthermore, based on the results predicted by the
RNAhybrid program, four putative microRNAs (miRNAs) were identified, and the
above SNPs were found to present in the seed region of these miRNAs. These
miRNAs have a differential binding affinity for each allele of SNPs
g.54853A>G, g.55441A>G, and g.55930C>T.
The in vivo evidence of intramuscular fat deposition in the LM tissue showed that
these SNPs affected the regulation of intramuscular fat deposition in Korean
cattle steer. Thus, the g.54853A>G, g.55441A>G, and
g.55930C>T could be considered as causal mutations regulating
intramuscular fat deposition in Korean cattle steer.
Introduction
Intramuscular fat deposition in the longissimus dorsi muscle (LM) of Korean
cattle is regulated by several genes related to lipid metabolic processes,
such as adipogenesis, lipogenesis, glycerolipid synthesis, and lipolysis.
Several previous studies have reported that by increasing the deposition of
intramuscular fat in Korean cattle steer, the expression levels of mRNAs
related to adipogenesis, glycerolipid synthesis, and lipogenesis were
upregulated, whereas those of mRNAs related to lipolysis were downregulated
(Jeong
et al., 2012; Kim et al., 2008). Among these metabolic processes, the mRNA
abundance of the glycerol-3-phosphate acyltransferase 1 (GPAT1), which catalyzes
the initial and committed step in glycerolipid biosynthesis, showed the
greatest correlation with intramuscular fat
content (Jeong
et al., 2012).
Bovine glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), also known as
GPAT1, is a member of the GPAT gene family and is an enzyme that synthesizes
lysophosphatidic acid (LPA) by transferring acyl groups to
glycerol-3-phosphate (Yu et al.,
2017). Thus, this enzyme catalyzes the initial and committed step in
glycerolipid biosynthesis and plays a key role in regulating the level of
cellular triacylglycerol in cattle
(Yu et al., 2017).
The GPAM gene is located on bovine chromosome 26q22 and is composed of 21 exons
and 20 introns. It is 3689 bp in length and has a much extended 3′-untranslated
region (3′-UTR), which is longer than that of the
other bovine genes. Furthermore, the 3′-UTR of GPAM contains a
polyadenylation signal site, erythroid 15-lipoxygenase differentiation
control elements (15-LOX-DICEs), and cytoplasmic polyadenylation elements
(CPEs) that affect the regulation of triacylglycerol synthesis. Roy et al. (2006)
reported that an extended 3′-UTR is significant for GPAM gene
regulation, and a cis-element might be implicated in mRNA stabilization and
translational control. The transmembrane domain of the GPAM protein is bound to
the outer membrane of mitochondria and its N- and C-termini are located in
the cytoplasm (Roy et al.,
2006).
Recently, Yu et al. (2017) reported that the knockdown of GPAM expression
significantly reduced the synthesis of triglycerides in bovine embryonic
fibroblast (BEF) cells, and the genetic variation of GPAM was significantly
associated with the fatty acid composition of intramuscular fat in cattle.
Furthermore, Roy et al. (2006) reported that the cis-regulatory elements
(15-LOX-DICEs and CPEs), which regulate the genes related to lipid
degradation and polyadenylation signal, respectively, are located in the
3′-UTR of the GPAM gene.
Therefore, the aim of this study was to identify SNPs related to the
regulation of glycerolipid synthesis in the 3′-UTR of GPAM and to verify
the function of SNPs affecting intramuscular fat deposition in Korean cattle
steer.
Materials and methodsAnimals, DNA extraction, SNP discovery
Animal welfare issue was followed according to approved guidelines of the
Animal Care and Use Committee of Hankyong National University. LM tissue
samples were collected from Korean cattle (n=192) raised in Pyeongchang
(Gangwon, Republic of Korea). The marbling grade of this beef was
classified according to the carcass grading standard of the Korea Institute
of Animal Products Quality Evaluation (KAPE, 2017). The KAPE provided grades
for beef marbling standard (BMS) score. All steers were maintained under
constant environmental conditions, with two types of commercial feeds in six
feedlots. Genomic DNA was extracted from the LM tissue using a
LaboPass™ tissue mini kit (Cosmo Genetech, Seoul,
Republic of Korea). In order to discover SNPs, the 3′-UTR sequence of GPAM was
obtained from the National Center for Biotechnology Information (NCBI)
GenBank database (accession no. NC_037353.1). The primers
were designed using NCBI Primer-BLAST based on the selected polymorphism
sites, and the primer sequences are shown in Table S1 in the Supplement. The
sequencing was performed according to a previous study
(Lee et al.,
2010), and SNPs were discovered using the “SNP Hunting” option of the
Sequencer v5.2.4 program (Gene Codes Corp., Ann Arbor, MI, USA). In order to map
the functional SNPs on DNA, mRNA (NM_001012282.1), and
protein (NP_01011282.1), these sequences were aligned using
the NCBI graphical sequence viewer. The correlation coefficients between SNP
pairs were calculated by Haploview 4.1 (Broad Institute, Cambridge, MA, USA)
with genotypes of the GPAM gene in Korean cattle.
Effect of SNPs in the 3′-UTR of the GPAM
gene on marbling scores in Korean cattle steer (n=190).
a,b Means with the same superscript in the same row for each
quality are not significantly different (p<0.05).
1 LSM: least square mean. 2 SE: standard error.
SNP genotyping and statistical analysis
SNPs were genotyped commercially using the Fluidigm®
SNP™-type assay platform according to
a previous study (Oh et al., 2018).
In order to evaluate the association SNPs and carcass traits, these data
were analyzed using a generalized linear model (GLM) in SPSS v22 (IBM,
Chicago, IL, USA) with the following equation:
Yijkl=μ+Farmi+Sirej+SNPk+βage+eijkl,
where Yijkl is the observed marbling score of Korean cattle; μ is the overall mean;
SNPk is the fixed effect of SNP genotype or haplotype;
Farmi is the fixed effect of the feed type in farm;
Sirej is the random
effect of the sire; βage is the covariation of age; and
eijkl is random error. The correlation coefficient between SNP pairs in the
3′-UTR of GPAM was analyzed using the Haploview program (Broad
Institute, USA). The relationship between SNPs and beef quality grades
(1++ and 2) was analyzed using Fisher's exact test in SPSS v22 (IBM,
USA).
Bioinformatics analysis of target microRNAs
In order to identify the microRNAs (miRNAs) that bind to candidate functional SNPs in
the 3′-UTR of GPAM, the TargetScan (http://www.targetscan.org,
last access: 15 November 2019) and
miRNA_Target (Kumar et al., 2012) software programs were
used. Subsequently, their sequences were obtained from miRBase
(http://www.mirbase.org, last access: 15 November 2019). The RNAhybrid program
(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid, last access: 15 November 2019) was employed to
calculate the minimum free energy (MFE) of binding between miRNAs and their
alleles, and the threshold was selected according to a previous
study (Knox et al., 2018; Rehmsmeier
et al., 2004).
ResultsIdentification of SNPs and their functional characterization
In the present study, we discovered 11 SNPs within the 3′-UTR of
GPAM in a Korean cattle steer population using the direct sequencing method. The
positions of SNPs within the 3′-UTR of GPAM and their pairwise
correlation coefficients are shown in Fig. 1. As shown in Fig. 1a,
although the detected 11 SNPs were present in the 3′-UTR of GPAM, they
were not specifically present in the sequences of four 15-LOX-DICEs and two
CPEs present in the 3′-UTR. For determining linkage disequilibrium
(LD) among 11 polymorphic SNPs in the 3′-UTR of GPAM, we calculated the
correlation coefficient between SNP pairs using the Haploview software. The
correlation coefficient between SNP pairs is shown in Fig. 1b. As shown in
Fig. 1b, two LD blocks were detected in the 3′-UTR of GPAM. The LD
block structure included two SNPs, whereas the other structure included four
SNPs. Three SNPs, g.54853A>G, g.55441A>G, and
g.55930C>T, were strongly correlated with each other, except for
the g.55517G>A SNP.
Positions of single-nucleotide polymorphisms (SNPs) in the
3′-UTR of the GPAM gene and their pairwise correlation.
(a) SNP position in the 3′-UTR of the GPAM gene. A total of 11 SNPs,
15-LOX-DICE and CPE, which are cis-regulatory elements, were mapped in the
3′-UTR of Korean cattle steer GPAM. 15-LOX-DICE and CPE are
represented by black and grey boxes, respectively. (b) Pairwise SNP
correlation. The color code on the Haploview plot follows the r2 color
scheme: white (r2=0); shades of grey (0<r2<1); black
(r2=1). The numbers in the cells are the
r2 values. However, the r2 value of 1.0 is not shown (empty cell).
Previous studies have reported that the 3′-UTR of the GPAM gene is
longer than the open reading frame (ORF) region and includes a cis-element
that plays a key role in mRNA stabilization and translational control
(Roy
et al., 2006; Yu et al., 2017). Furthermore, Yu et al. (2017) showed that the
SNPs in the 3′-UTR of GPAM were significantly associated with fatty acid
composition of intramuscular fat and marbling score in a beef cattle
population.
Association of functional SNPs with marbling score
In order to evaluate the function of 11 SNPs in the regulation of
glycerolipid synthesis, we analyzed the association between these SNPs and
marbling score. The effects of these SNPs and their combinations on the
marbling score of Korean cattle are shown in Tables 1 and 2.
In vivo evidence of the
genotype effect of functional SNPs in the 3′-UTR of GPAM on
the beef quality in Korean cattle steer. (a) Association of candidate
functional SNPs with Korean cattle steer beef quality grade. (b) Grade system
of beef quality.
As shown in Table 1, four SNPs (g.54316A>C, g.54853A>G,
g.55441A>G, and g.55930C>T) were significantly
associated with the marbling score of the Korean cattle steer population.
The marbling scores in group with the heterozygote genotypes of SNPs
g.54316A>C, g.54853A>G, g.55441A>G, and
g.55930C>T were significantly higher than those with the
homozygote genotypes of these SNPs. Furthermore, as shown in Table 2, the
combination types of SNPs g.54853A>G, g.55441A>G,
and g.55930C>T were significantly associated with the
marbling score of the Korean cattle steer population. Especially, the group
with the combination types GA, GA, and TC had the highest average marbling
score in the studied Korean cattle steer population.
Effect of combination SNPs on marbling scores in Korean
cattle steer (n=189).
BlockCombinationNo. ofMarblingp valuetypeanimalsLSM ± SEBlock 1CC-AA1406.129 ± 0.1760.918CT-AT416.355 ± 0.302CT-AA37.000 ± 0.960TT-TT55.400 ± 0.743Block 2GG-GG-TT606.019 ± 0.2690.044GG-GG-TC16.000 ± 1.749GA-GA-TC776.433 ± 0.234AA-GA-CC15.000 ± 1.749AA-AA-TC16.000 ± 1.749AA-AA-CC495.628 ± 0.295miRNA prediction and its target alteration by SNP alleles
In the present study, we predicted miRNAs that bind to candidate functional
SNPs using a bioinformatics tool, in order to determine whether these SNPs
had an effect on the regulation of glycerolipid synthesis. We identified
four miRNAs that bind to candidate functional SNPs present in the seed
region of miRNA using the online software programs TargetScan and
miRNA_Target (Table 3). To increase the credibility of our
results, we used the RNAhybrid software to quantitatively determine the
binding affinity between miRNAs and SNPs located in the seed region.
Information of miRNAs that bind on candidate
functional SNPs in the 3′-UTR of the
GPAM gene.
SNPAllele type MAF11miRNAMFE22 (kcal mol-1) Seed region typeMajorMinorMajorMinorg.54853A>GGA0.474bta-miR-2418-21.37mer-m8g.55441A>GGA0.471bta-miR-375-18.97mer-m8bta-miR-2479-11.97mer-A1g.55930C>TTC0.474bta-miR-2468-19.87mer-m8
1 MAF: minor allele frequency. 2 MFE: minimum free energy.
As shown in Table 3, three SNPs (g.54853A>G,
g.55441A>G, and g.55930C>T) were predicted to bind
to four miRNAs (bta-miR-2418, bta-miR-375, bta-miR-2479, and bta-miR-2468)
according to the seed region type. The seed region or seed sequence, which
is two to seven nucleotides long at the 5′ end of the miRNA sequence, is
essential for the binding of the partially complementary miRNA to the mRNA.
The seed region is classified into atypical sites, canonical sites and
marginal depending on the number of nucleotides matching between the seed
sequence and the mRNA (Bartel, 2009). Another important
factor on which miRNA binding depends is whether is adenine or guanine is
the first nucleotide at the 5′ end of
miRNA (Bartel, 2009). In addition, the binding
efficiency is in the order of (a) 8mer, (b) 7mer-m8, (c) 7mer-A1, and (d) 6mer. As
shown in Table 3, the seed region types of the miRNAs bta-miR-2418,
bta-miR-375, and bta-miR-2468 were all 7mer-m8, except for bta-miR-2479.
Especially, to increase the binding efficiency, we analyzed the G-U wobble
base pair parameter using the RNAhybrid software. The binding energies of
the miRNAs that bind to the sites with major alleles of the three SNPs were
all zero. As a result of the minor alleles shown in Table 3, the binding
energy of the miRNA-mRNA seed region with an A allele of SNP
g.54853A>G was the lowest at -21.3 kcal mol-1, whereas the
binding energy between the A allele of SNP g.55441A>G and
miRNA bta-miR-2479 was the highest at -11.9 kcal mol-1.
<italic>In vivo</italic> evidence of the genotype effect of functional SNPs on marbling score
To validate the effect of these functional SNPs on intramuscular fat
deposition in vivo, we determined the relationship between three functional SNPs
and Korean cattle beef grades (Fig. 2). As shown in Fig. 2, three
candidate functional SNPs (g.54853A>G, g.55441A>G,
and g.55930C>T) caused a significant difference between beef
grades “1++” and “2”. As shown in Table 3, the binding affinities
of the minor alleles of candidate SNPs were higher than those of their major
alleles. Thus, these results suggested that the miRNA-minor alleles of these
SNPs act as inhibitors in the regulation of glycerolipid synthesis.
As a result of the relationship between these SNPs and beef grades, as shown
in Fig. 2, we identified that the “2” beef grade in the group with the
minor alleles of these SNPs was significantly lower than that in the group
with major alleles (p<0.05).
Discussion
In the present study, we evaluated whether the 3′-UTR of GPAM was
influenced by the regulation of glycerolipid synthesis. The 3′-UTR
of GPAM is 3689 bp in length and is longer than those of the other genes
(Chen et al., 2012). The 3′-UTR has an
important influence on the regulation of gene expression because a potential
miRNA response element (MRE) is present in this region
(Arnold et
al., 2012).
Recently, Yu et al. (2017) reported that an SNP in the 3′-UTR of
GPAM was significantly associated with fatty acid composition of intramuscular
fat and marbling score in a beef cattle population. In the present results,
as shown in Tables 1 and 2, we demonstrated that SNPs
g.54316A>C, g.54853A>G, g.55441A>G, and
g.55930C>T, which are located in the 3′-UTR, were
associated with marbling score in the studied Korean cattle steer
population. These results coincide with those reported by Yu et al. (2017).
Furthermore, as in vivo evidence of the genotype effect of functional SNPs on
marbling score, the group with the major alleles of these SNPs had
significantly lower binding affinities between the seed region allele and
miRNA than those with the minor alleles of these SNP. Thus, these results
suggested that these SNPs had an important effect on the regulation of
glycerolipid synthesis in the studied Korean cattle steer population.
We found that the 3′-UTR of the GPAM gene contained four 15-LOX-DICEs
and two CPEs, which regulate mRNA translation and structure stabilization.
However, no SNPs were detected in the region containing four 15-LOX-DICEs
and two CPEs. In cattle, miRNA targeting has predominantly been associated
with the 3′-UTR region of the transcripts derived from ORFs,
typically leading to down-regulation through triggering RNA degradation, RNA
instability, and/or reduction (He and
Hannon, 2004; Li et al., 2011). Thus, as shown in Table 3, four miRNAs were
predicted to bind to the seed region including SNPs g.54853A>G,
g.55441A>G, and g.55930C>T, using the software
programs TargetScan and RNAhybrid. According to the minimum free energy of
the binding affinity, each allele of these SNPs had a different binding
affinity. Furthermore, this difference in binding affinities led to a
significant difference between beef grades “1++” and “2”. Thus,
our results suggested that the three SNPs, which are present in the 3′-UTR,
could be genetic variations influencing the regulation of GPAM gene
expression.
Conclusions
Glycerol-3-phosphate acyltransferase, mitochondrial, the enzyme located on
the mitochondrial outer membrane, catalyzes the initial and committed step
in glycerolipid synthesis in the lipid metabolism of cattle. In the present
study, we determined whether the SNPs located in the 3′-UTR of
GPAM affect the regulation of gene expression. Of the 11 SNPs detected in the
3′-UTR of GPAM, three SNPs, g.54316A>C, g.54853A>G,
g.55441A>G, and g.55930C>T, were significantly
associated with marbling score in Korean cattle steer population and showed
strong pairwise correlation. Furthermore, we identified four putative miRNAs
and found that these SNPs were present in the seed region of these miRNAs.
These miRNAs showed a differential binding affinity for each allele of
SNPs g.54316A>C, g.54853A>G, g.55441A>G,
and g.55930C>T, leading to a significant difference between
beef quality grades “1++” and “2”. Thus, our results suggested that
three SNPs in the 3′-UTR could be genetic variations affecting the
regulation of GPAM gene expression.
Data availability
The original data of the paper are available from
the corresponding author upon request.
The supplement related to this article is available online at: https://doi.org/10.5194/aab-64-27-2021-supplement.
Author contributions
HK and DYO performed the data analyses and wrote the manuscript. YL revised the manuscript and designed the experiment. All authors reviewed and approved the final paper.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by a research grant from Hankyong National
University in 2017.
Financial support
This research has been supported by a grant from Hankyong National University (grant no. 2017-056).
Review statement
This paper was edited by Steffen Maak and reviewed by Jae-Sung Lee and two anonymous referees.
ReferencesArnold, M., Ellwanger, D. C., Hartsperger, M. L., Pfeufer, A., and
Stümpflen, V.: Cis-acting polymorphisms affect complex traits through
modifications of MicroRNA regulation pathways, Plos One, 7, 1–12,
10.1371/journal.pone.0036694, 2012.Bartel, D. P.: MicroRNAs: Target Recognition and Regulatory Functions, Cell,
136, 215–233, 10.1016/j.cell.2009.01.002, 2009.Chen, C. Y., Chen, S. T., Juan, H. F., and Huang, H. C.: Lengthening of 3'UTR
increases with morphological complexity in animal evolution, Bioinformatics,
28, 3178–3181, 10.1093/bioinformatics/bts623, 2012.He, L. and Hannon, G. J.: MicroRNAs: Small RNAs with a big role in gene
regulation, Nat. Rev. Genet., 5, 522–531, 10.1038/nrg1379, 2004.Jeong, J., Kwon, E. G., Im, S. K., Seo, K. S., and Baik, M.: Expression of
fat deposition and fat removal genes is associated with intramuscular fat
content in longissimus dorsi muscle of Korean cattle steers, J. Anim. Sci.,
90, 2044–2053, 10.2527/jas.2011-4753, 2012.Kim, N., Kim, S., Heo, K., Yoon, D., Lee, C., Im, S., and Park, E.:
Expression Profiles of Triacylglycerol Biosynthesis Genes on Fattening
Stages in Hanwoo, J. Anim. Sci. Technol., 50, 293–300,
10.5187/jast.2008.50.3.293, 2008 (in Korean).Knox, B., Wang, Y., Rogers, L. J., Xuan, J., Yu, D., Guan, H., Chen, J.,
Shi, T., Ning, B., and Kadlubar, S. A.: A functional SNP in the 3'-UTR of
TAP2 gene interacts with microRNA hsa-miR-1270 to suppress the gene
expression, Environ. Mol. Mutagen., 59, 134–143, 10.1002/em.22159,
2018.Korea Institute of Animal Products Quality Evaluation [KAPE], available at: http://www.ekapepia.or.kr/view/eng/system/beef.asp, last access: 1 February 2017.Kumar, A., Wong, A. K-L, Tizard, M. L, Moore, R. J., and Lefèvre, C.: miRNA_Targets: a database for miRNA target predictions in coding and non-coding regions of mRNAs, Genomics., 100, 352–356, 10.1016/j.ygeno.2012.08.006, 2012.
Lee, Y. S., Oh, D. Y., Kim, J. J., Lee, J. H., Park, H. S., and Yeo, J. S.: A
single nucleotide polymorphism in LOC534614 as an unknown gene associated
with body weight and cold carcass weight in Hanwoo (Korean Cattle),
Asian-Austral. J. Anim., 23, 1543–1551, 10.5713/ajas.2010.10113, 2010.Li, H., Zhang, Z., Zhou, X., Wang, Z., Wang, G., and Han, Z.: Effects of
MicroRNA-143 in the differentiation and proliferation of bovine
intramuscular preadipocytes, Mol. Biol. Rep., 38, 4273–4280,
10.1007/s11033-010-0550-z, 2011.Oh, D.-Y., Nam, I., Hwang, S., Kong, H., Lee, H., Ha, J., Baik, M., Oh, M.
H., Kim, S., Han, K., and Lee, Y.: In vivo evidence on the functional
variation within fatty acid synthase gene associated with lipid metabolism
in bovine longissimus dorsi muscle tissue, Genes Genom., 40,
289–294, 10.1007/s13258-017-0634-4, 2018.Rehmsmeier, M., Steffen, P., Hoechsmann, M., and Giegerich, R.: Fast and
effective prediction of microRNA/target duplexes RNA, RNA, available
at: http://rnajournal.cshlp.org/content/10/10/1507.abstract (last access: 15 November 2019), 2004.Roy, R., Ordovas, L., Taourit, S., Zaragoza, P., Eggen, A., and Rodellar, C.:
Genomic structure and an alternative transcript of bovine mitochondrial
glycerol-3-phosphate acyltransferase gene (GPAM), Cytogenet. Genome Res.,
112, 82–89, 10.1159/000087517, 2006.Yu, H., Zhao, Z., Yu, X., Li, J., Lu, C., and Yang, R.: Bovine lipid
metabolism related gene GPAM: Molecular characterization, function
identification, and association analysis with fat deposition traits, Gene,
609, 9–18, 10.1016/j.gene.2017.01.031, 2017.