Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.Cilia are microtubule-based organelles that function in motility, sensory reception, and signaling (1). Ciliary dysfunction results in numerous diseases and disorders commonly known as ciliopathies. Intraflagellar transport (IFT) is involved in cilium formation (2, 3) but also functions in other cellular processes, such as the recycling of Tcell receptors at the immune synapse (4). IFT relies on kinesin-2 and IFT-dynein molecular motors moving along the microtubule-based axoneme of cilia (5-7) and on the IFT complex, which contains at least 20 different protein subunits. Although ~600 proteins are known to reside in the cilium (8), we know very little about how they are recognized as ciliary cargo by the IFT machinery (9-11).To identify potential cargo-binding sites on the IFT complex, we carried out bioinformatical and biochemical screening and identified conserved domains that were not required for IFT complex formation. We reasoned that such domains could protrude from the IFT particlecore structure and would thus be in a prime position for cargo recognition. The two IFT core (12). Given that the cilium consists of a MT-based axoneme, IFT of large quantities of tubulin is required for cilium formation (13). We thus tested the tubulin-binding properties of HsIFT81N using affinity pull-downs ( Fig. 1D and fig. S4E) and microscale thermophoresis (MST) with unpolymerized bovine αβ-tubulin (Fig. 1, E S5F). Thus, the tubulin-binding module is formed by the IFT74/81 complex rather than by IFT81N alone.To dissect the binding mode in the IFT74/81:αβ-tubulin complex, samples were prepared from MT and unpolymerized αβ-tubulin lacking the highly acidic C-terminal tails, often referred to as E-hooks (12) ( fig. S5A). αβ-tubulin lacking E-hooks had similar affinity for IFT81N as intact tubulin ( fig. S5, B and C), which suggested that IFT81N recognizes the globular domain of αβ-tubulin with no substantial interaction with the E-hooks. IFT74/81 displayed robust MT binding in sedimentation assays, which was, however, reduced to background levels in the absence of the β-tubulin E-hook ( fig. S5E). Thus, IFT81N appears to bind the globular domain of tubulin to provide specificity, and IFT74N recognizes the β-tubulin tail to increase affinity (Fig. 1H).To examine the role of tubulin binding by IFT74/81 in a cellular system, we transiently expressed Flag-HsIFT81 or Flag-HsIFT81ΔN in human RPE-1 cells and induced formation ...

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

Improvement in feed conversion efficiency can improve the sustainability of beef cattle production, but genomic selection for feed efficiency affects many underlying molecular networks and physiological traits. This study describes the differences between steer progeny of two influential Angus bulls with divergent genomic predictions for residual feed intake (RFI). Eight steer progeny of each sire were phenotyped for growth and feed intake from 8 mo. of age (average BW 254 kg, with a mean difference between sire groups of 4.8 kg) until slaughter at 14–16 mo. of age (average BW 534 kg, sire group difference of 28.8 kg). Terminal samples from pituitary gland, skeletal muscle, liver, adipose, and duodenum were collected from each steer for transcriptome sequencing. Gene expression networks were derived using partial correlation and information theory (PCIT), including differentially expressed (DE) genes, tissue specific (TS) genes, transcription factors (TF), and genes associated with RFI from a genome-wide association study (GWAS). Relative to progeny of the high RFI sire, progeny of the low RFI sire had -0.56 kg/d finishing period RFI (P = 0.05), -1.08 finishing period feed conversion ratio (P = 0.01), +3.3 kg^0.75 finishing period metabolic mid-weight (MMW; P = 0.04), +28.8 kg final body weight (P = 0.01), -12.9 feed bunk visits per day (P = 0.02) with +0.60 min/visit duration (P = 0.01), and +0.0045 carcass specific gravity (weight in air/weight in air—weight in water, a predictor of carcass fat content; P = 0.03). RNA-seq identified 633 DE genes between sire groups among 17,016 expressed genes. PCIT analysis identified >115,000 significant co-expression correlations between genes and 25 TF hubs, i.e. controllers of clusters of DE, TS, and GWAS SNP genes. Pathway analysis suggests low RFI bull progeny possess heightened gut inflammation and reduced fat deposition. This multi-omics analysis shows how differences in RFI genomic breeding values can impact other traits and gene co-expression networks.

Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.

Two high-density single nucleotide polymorphism (SNP) genotyping arrays have recently become available for bovine genomic analyses, the Illumina High-Density Bovine BeadChip Array (777,962 SNP) and the Affymetrix Axiom Genome-Wide BOS 1 Array (648,874 SNP). These products each have unique design and chemistry attributes, and the extent of marker overlap and their potential utility for quantitative trait loci fine mapping, detection of copy number variation, and multibreed genomic selection are of significant interest to the cattle community. This is the first study to compare the performance of these 2 arrays. Deoxyribonucleic acid samples from 16 dairy cattle (10 Holstein, 6 Jersey) were used for the comparison. An independent set of DNA samples taken from 46 Jersey cattle and 18 Holstein cattle were used to ascertain the amount of SNP variation accounted by the 16 experimental samples. Data were analyzed with SVS7 software (Golden Helix Inc., Bozeman, MT) to remove SNP having a call rate less than 90%, and linkage disequilibrium pruning was used to remove linked SNP (r² ≥ 0.9). Maximum, average, and median gaps were calculated for each analysis based on genomic position of SNP on the bovine UMD3.1 genome assembly. All samples were successfully genotyped (≥ 98% SNP genotyped) with both platforms. The average number of genotyped SNP in the Illumina platform was 775,681 and 637,249 for the Affymetrix platform. Based on genomic position, a total of 107,896 SNP were shared between the 2 platforms; however, based on genotype concordance, only 96,031 SNP had complete concordance at these loci. Both Affymetrix BOS 1 and Illumina BovineHD genotyping platforms are well designed and provide high-quality genotypes and similar coverage of informative SNP. Despite fewer total SNP on BOS 1, 19% more SNP remained after linkage disequilibrium pruning, resulting in a smaller gap size (5.2 vs. 6.9 kb) in Holstein and Jersey samples relative to BovineHD. However, only 224,115 Illumina and 241,038 Affymetrix SNP remained following removal of SNP with a minor allele frequency of zero in Holstein and Jersey samples, resulting in an average gap size of 11,887 bp and 11,018 bp, respectively. Combining the 354,348 informative (r² ≥ 0.9), polymorphic (minor allele frequency ≥ 0), unique SNP data from both platforms decreased the average gap size to 7,560 bp. Genome-wide copy number variant analyses were performed using intensity files from both platforms. The BovineHD platform provided an advantage to the copy number variant data compared with the BOS 1 because of the larger number of SNP, higher intensity signals, and lower background effects. The combined use of both platforms significantly improved coverage over either platform alone and decreased the gap size between SNP, providing a valuable tool for fine mapping quantitative trait loci and multibreed animal evaluation.

Genomic selection involves the assessment of genetic merit through prediction equations that allocate genetic variation with dense marker genotypes. It has the potential to provide accurate breeding values for selection candidates at an early age and facilitate selection for expensive or difficult to measure traits. Accurate across-breed prediction would allow genomic selection to be applied on a larger scale in the beef industry, but the limited availability of large populations for the development of prediction equations has delayed researchers from providing genomic predictions that are accurate across multiple beef breeds. In this study, the accuracy of genomic predictions for 6 growth and carcass traits were derived and evaluated using 2 multibreed beef cattle populations: 3,358 crossbred cattle of the U.S. Meat Animal Research Center Germplasm Evaluation Program (USMARC_GPE) and 1,834 high accuracy bull sires of the 2,000 Bull Project (2000_BULL) representing influential breeds in the U.S. beef cattle industry. The 2000_BULL EPD were deregressed, scaled, and weighted to adjust for between- and within-breed heterogeneous variance before use in training and validation. Molecular breeding values (MBV) trained in each multibreed population and in Angus and Hereford purebred sires of 2000_BULL were derived using the GenSel BayesCπ function (Fernando and Garrick, 2009) and cross-validated. Less than 10% of large effect loci were shared between prediction equations trained on (USMARC_GPE) relative to 2000_BULL although locus effects were moderately to highly correlated for most traits and the traits themselves were highly correlated between populations. Prediction of MBV accuracy was low and variable between populations. For growth traits, MBV accounted for up to 18% of genetic variation in a pooled, multibreed analysis and up to 28% in single breeds. For carcass traits, MBV explained up to 8% of genetic variation in a pooled, multibreed analysis and up to 42% in single breeds. Prediction equations trained in multibreed populations were more accurate for Angus and Hereford subpopulations because those were the breeds most highly represented in the training populations. Accuracies were less for prediction equations trained in a single breed due to the smaller number of records derived from a single breed in the training populations.

Cilia are microtubule-based hair-like structures that project from the surfaces of eukaryotic cells. Cilium formation relies on intraflagellar transport (IFT) to move ciliary proteins such as tubulin from the site of synthesis in the cell body to the site of function in the cilium. A large protein complex (the IFT complex) is believed to mediate interactions between cargoes and the molecular motors that walk along axonemal microtubules between the ciliary base and tip. A recent study using purified IFT complexes has identified a tubulin-binding module in the two core IFT proteins IFT74 and IFT81 that likely serves to bind and transport tubulin within cilia. Here, we calculate the amount of tubulin required to support the observed cilium assembly kinetics and explore the possibility of multiple tubulin binding sites within the IFT complex.

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H2O2 production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H2O2 production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H2O2 production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.