The Molecular Effects of a Polymorphism in the 5′UTR of Solute Carrier Family 44, Member 5 that Is Associated with Birth Weight in Holsteins

<div><p>Dystocia is a major problem for the dairy cattle industry, and the observed high rates of this condition stem from genetic selection to increase subsequent milk production of the calving female. Because smaller birth size does not adversely affect subsequent milk production, selecting for cows with a smaller birth size would reduce dystocia rates and be beneficial for both the cattle and the farmers. To identify genes that regulate birth weight, we conducted a genome-wide association study using 1151 microsatellite markers and identified a single nucleotide polymorphism (SNP) associated with birth weight: A-326G in the 5′ untranslated region (UTR) of <em>solute carrier family 44, member 5</em> (<em>SLC44A5</em>). Cows with higher birth weights carried the A polymorphism in the <em>SLC44A5</em> 5′ UTR, and the presence of the A polymorphism correlated with a high rate of dystocia. Luciferase assays and quantitative polymerase chain reaction (QPCR) assays revealed that <em>SLC44A5</em> transcripts with the A polymorphism are expressed at lower levels than those carrying the G polymorphism. <em>SLC44A5</em> encodes a choline transporter-like protein, and choline is a component of the major phospholipids of cell membranes. Uptake studies in HeLa cells demonstrated that <em>SLC44A5</em> knockdown reduces choline efflux, whereas <em>SLC44A5</em> overexpression resulted in the opposite effect. Furthermore, cell viability assays indicated that <em>SLC44A5</em> knockdown increased cell proliferation, whereas <em>SLC44A5</em> overexpression repressed proliferation. Taken together, our results suggest that calves with reduced <em>SLC44A5</em> expression are larger due to enhanced cell proliferation. This study provides novel insights into the molecular mechanisms that control birth weight in Holsteins and suggests that SLC44A5 may serve as a potential target for preventing dystocia.</p> </div

The <i>SLC44A5</i> 5′ UTR SNP controls its expression level.

<p>A. The relative luciferase activity of the 5′ UTR region of <i>SLC44A5</i>. The data are presented as the mean ± SEM. The <i>p</i>-value was calculated using the Student's t-test. B. A gel mobility shift assay of HeLa nuclear protein using the 5′ UTR region of <i>SLC44A5</i> as the probe. The binding indicated by the arrow was abolished by co-incubation with an unlabeled competitor with the A or G polymorphisms but not by a competitor containing the AP1 sequence (N). The G polymorphism-specific binding is indicated by the arrow with a star and was abolished only by co-incubation with an unlabeled competitor with the G polymorphism. C. The relative expression levels of <i>SLC44A5</i> in bovine tissues. D. The relative expression level of <i>SLC44A5</i> in the bovine brain. The data are presented as the mean ± SEM. The <i>p</i>-value was calculated using the Student's t-test. E. Average allele-specific expression level ± SE in the heterozygous bovine brain. The ratios of G to A relative to genomic DNA were shown.</p

A list of <i>SLC44A5</i> primers.

<p>A list of <i>SLC44A5</i> primers.</p

SLC44A5 suppresses proliferation.

<p>A. The relative proliferation of transfected HeLa cells. The data are presented as the mean ± SEM (n = 8). The <i>p</i>-values were calculated using the Student's t-test. B. The relative expression of genes related to proliferation and apoptosis in transfected HeLa cells. Each QPCR was performed with nine replicate with samples from three transfection experiments. The data are presented as the mean ± SEM. The <i>p</i>-values were calculated using the Student's t-test. *, **, and *** indicate <i>p</i><0.05, <i>p</i><0.005, and <i>p</i><0.0005, respectively.</p

The <i>SLC44A5</i> 5′ UTR SNP is associated with birth weight.

<p>A. The LD block for the critical region in chromosome 3. The numbers represent the positions of the microsatellite markers in bp. ACADM and SLC44A5 indicate the SNPs identified in each gene. B. The average birth weight ± SE values for the calves. The <i>p</i>-value was calculated using the Student's t-test. C. The average dystocia rate ± SE values for sires. The <i>p</i>-value was calculated by the Student's t-test.</p

A list of <i>ACADM</i> primers.

<p>A list of <i>ACADM</i> primers.</p

Birth weight is associated with a locus on chromosome 3.

<p>A. The distribution of birth weights among the samples. B, C, D. The association signals with birth weight for the 1^st (B), 2^nd (C), and 3^rd (D) screenings. The blue and red lines represent the threshold for chromosome-wide and genome-wide significance following the Bonferroni correction for multiple comparisons, respectively. E. A schematic representation of the genes (black arrow) and the microsatellite markers (blue dot) located in the critical region. The red dots represent the most significantly associated microsatellite markers. The numbers represent the positions of microsatellite markers in base pairs (bp).</p

Additional file 2: Table S1. of Impact of QTL minor allele frequency on genomic evaluation using real genotype data and simulated phenotypes in Japanese Black cattle

Number of SNPs before and after weighting for Speed's genomic relationship matrix. Table S2. Comparison of two and five minor allele frequency (MAF) categories. (DOC 62 kb

Additional file 1: Figure S1. of Impact of QTL minor allele frequency on genomic evaluation using real genotype data and simulated phenotypes in Japanese Black cattle

Distribution of progenies per sire in this population. The x-axis indicates the number of progenies per sire, and the y-axis represents the number of sires. Figure S2. QTL effect and QTL variance as a function of minor allele frequency (MAF). The x-axis indicates the MAF of SNPs, and the y-axis represents the QTL effect (a) and QTL variance (b) in a randomly selected replica. The results of varying distributions of QTL allele substitution effects (Gamma, gamma distribution model; EquV, equal variance model) for all MAF, QTL heritability (0.40), and the number of QTLs (500) are shown. (PPTX 51 kb

SMAP2 Regulates Retrograde Transport from Recycling Endosomes to the Golgi

<div><p>Retrograde transport is where proteins and lipids are transported back from the plasma membrane (PM) and endosomes to the Golgi, and crucial for a diverse range of cellular functions. Recycling endosomes (REs) serve as a sorting station for the retrograde transport and we recently identified evection-2, an RE protein with a pleckstrin homology (PH) domain, as an essential factor of this pathway. How evection-2 regulates retrograde transport from REs to the Golgi is not well understood. Here, we report that evection-2 binds to SMAP2, an Arf GTPase-activating protein. Endogenous SMAP2 localized mostly in REs and to a lesser extent, the trans-Golgi network (TGN). SMAP2 binds evection-2, and the RE localization of SMAP2 was abolished in cells depleted of evection-2. Knockdown of SMAP2, like that of evection-2, impaired the retrograde transport of cholera toxin B subunit (CTxB) from REs. These findings suggest that evection-2 recruits SMAP2 to REs, thereby regulating the retrograde transport of CTxB from REs to the Golgi.</p> </div