Comprehensive review on lipid metabolism and RNA methylation: Biological mechanisms, perspectives and challenges.

Adipose tissue plays a crucial role in maintaining energy balance, regulating hormones, and promoting metabolic health. To address disorders related to obesity and develop effective therapies, it is essential to have a deep understanding of adipose tissue biology. In recent years, RNA methylation has emerged as a significant epigenetic modification involved in various cellular functions and metabolic pathways. Particularly in the realm of adipogenesis and lipid metabolism, extensive research is ongoing to uncover the mechanisms and functional importance of RNA methylation. Increasing evidence suggests that RNA methylation plays a regulatory role in adipocyte development, metabolism, and lipid utilization across different organs. This comprehensive review aims to provide an overview of common RNA methylation modifications, their occurrences, and regulatory mechanisms, focusing specifically on their intricate connections to fat metabolism. Additionally, we discuss the research methodologies used in studying RNA methylation and highlight relevant databases that can aid researchers in this rapidly advancing field.

A chromosome-level genome of Chenghua pig provides new insights into the domestication and local adaptation of pigs.

As a country with abundant genetic resources of pigs, the domestication history of pigs in China and the adaptive evolution of Chinese pig breeds at different latitudes have rarely been elucidated at the genome-wide level. To fill this gap, we first assembled a high-quality chromosome-level genome of the Chenghua pig and used it as a benchmark to analyse the genomes of 272 samples from three genera of three continents. The divergence of the three species belonging to three genera, Phacochoerus africanus, Potamochoerus porcus, and Sus scrofa, was assessed. The introgression of pig breeds redefined that the migration routes were basically from southern China to central and southwestern China, then spread to eastern China, arrived in northern China, and finally reached Europe. The domestication of pigs in China occurred ∼12,000 years ago, earlier than the available Chinese archaeological domestication evidence. In addition, FBN1 and NR6A1 were identified in our study as candidate genes related to extreme skin thickness differences in Eurasian pig breeds and adaptive evolution at different latitudes in Chinese pig breeds, respectively. Our study provides a new resource for the pig genomic pool and refines our understanding of pig genetic diversity, domestication, migration, and adaptive evolution at different latitudes.

The role of mitochondrial dynamics and mitophagy in skeletal muscle atrophy: from molecular mechanisms to therapeutic insights.

Skeletal muscle is the largest metabolic organ of the human body. Maintaining the best quality control and functional integrity of mitochondria is essential for the health of skeletal muscle. However, mitochondrial dysfunction characterized by mitochondrial dynamic imbalance and mitophagy disruption can lead to varying degrees of muscle atrophy, but the underlying mechanism of action is still unclear. Although mitochondrial dynamics and mitophagy are two different mitochondrial quality control mechanisms, a large amount of evidence has indicated that they are interrelated and mutually regulated. The former maintains the balance of the mitochondrial network, eliminates damaged or aged mitochondria, and enables cells to survive normally. The latter degrades damaged or aged mitochondria through the lysosomal pathway, ensuring cellular functional health and metabolic homeostasis. Skeletal muscle atrophy is considered an urgent global health issue. Understanding and gaining knowledge about muscle atrophy caused by mitochondrial dysfunction, particularly focusing on mitochondrial dynamics and mitochondrial autophagy, can greatly contribute to the prevention and treatment of muscle atrophy. In this review, we critically summarize the recent research progress on mitochondrial dynamics and mitophagy in skeletal muscle atrophy, and expound on the intrinsic molecular mechanism of skeletal muscle atrophy caused by mitochondrial dynamics and mitophagy. Importantly, we emphasize the potential of targeting mitochondrial dynamics and mitophagy as therapeutic strategies for the prevention and treatment of muscle atrophy, including pharmacological treatment and exercise therapy, and summarize effective methods for the treatment of skeletal muscle atrophy.

Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

Dynamic changes in the transcriptome of tRNA-derived small RNAs related with fat metabolism.

The prevalence of obesity and overweight is steadily rising, posing a significant global challenge for humanity. The fundamental cause of obesity and overweight lies in the abnormal accumulation of adipose tissue. While numerous regulatory factors related to fat deposition have been identified in previous studies, a considerable number of regulatory mechanisms remain unknown. tRNA-derived small RNAs (tsRNAs), a novel class of non-coding RNAs, have emerged as significant regulators in various biological processes. In this study, we obtained small RNA sequencing data from subcutaneous white adipose tissue and omental white adipose tissue of lean and obese pigs. In addition, we similarly obtained tsRNAs profiles from scapular brown adipose tissue (BAT), inguinal white adipose tissue (iWAT) and epigonadal white adipose tissue (eWAT) of normal mice. Finally, we successfully identified a large number of expressed tsRNAs in each tissue type and identified tsRNAs conserved in different adipose tissues of pigs and mice. These datasets will be a valuable resource for elucidating the epigenetic mechanisms of fat deposition.

Short-term dietary choline supplementation alters the gut microbiota and liver metabolism of finishing pigs.

Choline is an essential nutrient for pig development and plays a role in the animal's growth performance, carcass characteristics, and reproduction aspects in weaned pigs and sows. However, the effect of choline on finishing pigs and its potential regulatory mechanism remains unclear. Here, we feed finishing pigs with 1% of the hydrochloride salt of choline, such as choline chloride (CHC), under a basic diet condition for a short period of time (14 days). A 14-day supplementation of CHC significantly increased final weight and carcass weight while having no effect on carcass length, average backfat, or eye muscle area compared with control pigs. Mechanically, CHC resulted in a significant alteration of gut microbiota composition in finishing pigs and a remarkably increased relative abundance of bacteria contributing to growth performance and health, including Prevotella, Ruminococcaceae, and Eubacterium. In addition, untargeted metabolomics analysis identified 84 differently abundant metabolites in the liver between CHC pigs and control pigs, of which most metabolites were mainly enriched in signaling pathways related to the improvement of growth, development, and health. Notably, there was no significant difference in the ability of oxidative stress resistance between the two groups, although increased bacteria and metabolites keeping balance in reactive oxygen species showed in finishing pigs after CHC supplementation. Taken together, our results suggest that a short-term supplementation of CHC contributes to increased body weight gain and carcass weight of finishing pigs, which may be involved in the regulation of gut microbiota and alterations of liver metabolism, providing new insights into the potential of choline-mediated gut microbiota/metabolites in improving growth performance, carcass characteristics, and health.

A Novel tRNA-Derived Fragment, tRFGlnCTG, Regulates Angiogenesis by Targeting Antxr1 mRNA.

As a novel non-coding RNA with important functions corresponding to various cellular stresses, the function of tRFs in angiogenesis remains unclear. Firstly, small RNA sequencing was performed on normal and post-muscle injury mouse tibialis anterior muscle to identify and analyse differentially expressed tRF/tiRNA. tRNA GlnCTG-derived fragments (tRFGlnCTG) were found to be overexpressed in high abundance in the damaged muscle. Subsequent in vitro experiments revealed that the overexpression of tRFGlnCTG suppressed the vascular endothelial cells' viability, cell cycle G1/S transition, proliferation, migration, and tube-formation capacity. Similarly, in vivo experiments showed that the tRFGlnCTG decreased the relative mRNA levels of vascular endothelial cell markers and pro-angiogenic factors and reduced the proportion of CD31-positive cells. Finally, luciferase activity analysis confirmed that the tRFGlnCTG directly targeted the 3'UTR of Antxr1, leading to a significant reduction in the mRNA expression of the target gene. These results suggest that tRFGlnCTG is a key regulator of vascular endothelial cell function. The results provide a new idea for further exploration of the molecular mechanisms that regulate angiogenesis.

miRNAs derived from cobra venom exosomes contribute to the cobra envenomation.

Currently, there is an increasing amount of evidence indicating that exosomes and the miRNAs they contain are crucial players in various biological processes. However, the role of exosomes and miRNAs in snake venom during the envenomation process remains largely unknown. In this study, fresh venom from Naja atra of different ages (2-month-old, 1-year-old, and 5-year-old) was collected, and exosomes were isolated through ultracentrifugation. The study found that exosomes with inactivated proteins and enzymes can still cause symptoms similar to cobra envenomation, indicating that substances other than proteins and enzymes in exosomes may also play an essential role in cobra envenomation. Furthermore, the expression profiles of isolated exosome miRNAs were analyzed. The study showed that a large number of miRNAs were co-expressed and abundant in cobra venom exosomes (CV-exosomes) of different ages, including miR-2904, which had high expression abundance and specific sequences. The specific miR-2094 derived from CV-exosomes (CV-exo-miR-2904) was overexpressed both in vitro and in vivo. As a result, CV-exo-miR-2904 induced symptoms similar to cobra envenomation in mice and caused liver damage, demonstrating that it plays a crucial role in cobra envenomation. These results reveal that CV-exosomes and the miRNAs they contain play a significant regulatory role in cobra envenomation. Our findings provide new insights for the treatment of cobra bites and the development of snake venom-based medicines.

The global perspective on peroxisome proliferator-activated receptor γ (PPARγ) in ectopic fat deposition: A review.

Excessive expansion of adipocytes can have unhealthy consequences as excess free fatty acids enter other tissues and cause ectopic fat deposition by resynthesizing triglycerides. This lipid accumulation in various tissues is harmful and can increase the risk of related metabolic diseases such as type II diabetes, cardiovascular disease, and insulin resistance. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that play a key role in energy metabolism as fatty acid metabolism sensors, and peroxisome proliferator-activated receptor γ (PPARγ) is the main subtype responsible for fat cell differentiation and adipogenesis. In this paper, we introduce the main structure and function of PPARγ and its regulatory role in the process of lipogenesis in the liver, kidney, skeletal muscle, and pancreas. This information can serve as a reference for further understanding the regulatory mechanisms and measures of the PPAR family in the process of ectopic fat deposition.

Comparison of Growth Performance and Plasma Metabolomics between Two Sire-Breeds of Pigs in China.

The Yorkshire pigs, renowned for their remarkable growth rate, low feed conversion ratio (FCR), and high meat production, emerge as a novel preference for paternal breeding. In this study, we found that purebred paternal Yorkshire pigs (PY) surpass the purebred Duroc breed in terms of growth rate. Specifically, purebred PY attain a weight of 100 kg at an earlier age compared to purebred Duroc (Male, 145.07 vs. 162.91; Female, 145.91 vs. 167.57; p-value < 0.01). Furthermore, different hybrid combinations suggest that offspring involving purebred PY exhibit superior growth performance. Compared with purebred Duroc, the offspring of purebred PY have an earlier age in days (173.23 vs. 183.54; p-value < 0.05) at the same slaughter weight. The changes of plasma metabolites of 60-day-old purebred boars in the two sire-breeds showed that 1335 metabolites in plasma were detected. Compared with Duroc, 28 metabolites were down-regulated and 49 metabolites were up-regulated in PY. Principal component analysis (PCA) discerned notable dissimilarities in plasma metabolites between the two sire-breeds of pigs. The levels of glycerol 3-phosphate choline, cytidine, guanine, and arachidonic acid increased significantly (p-value < 0.05), exerting an impact on their growth and development. According to our results, PY could be a new paternal option as a terminal sire in three-way cross system.

A Comparative Study on the Growth Performance and Gut Microbial Composition of Duroc and Yorkshire Boars.

The intestinal microbiota is required for maintaining the development and health of the host. However, the gut microbiota contributing to the regulation of the growth performance and health of Duroc and Yorkshire boars remains largely unknown. In this study, we first evaluated the difference in the growth performance between Duroc and Yorkshire boars when their body weight reached 100 kg. Relative to Duroc boars, Yorkshire boars weighed 100 kg at a younger age and exhibited a significantly lower backfat thickness and eye muscle area. Microbial analysis of the fecal samples revealed a marked difference in gut microbiota composition between the two pig models and remarkably increased α-diversity in Yorkshire boars compared to Duroc boars. Further analysis indicated that Bacteroidota, Prevotellaceae, and Ruminococcaceae might be associated with the growth performance and lean meat rate of Yorkshire boars. Taken together, these results highlight that Yorkshire boars exhibit a faster growth rate and higher lean meat rate compared to Duroc boars, and these differences may be attributed to the influence of the gut microbiota, thereby providing valuable insight into optimizing pig breeding systems and selecting terminal paternal sires to enhance overall productivity and quality.

Effects of dietary L-Citrulline supplementation on growth performance, meat quality, and fecal microbial composition in finishing pigs.

Gut microbiota play an important role in the gut ecology and development of pigs, which is always regulated by nutrients. This study investigated the effect of L-Citrulline on growth performance, carcass characteristics, and its potential regulatory mechanism. The results showed that 1% dietary L-Citrulline supplementation for 52 days significantly increased final weight, liveweight gain, carcass weight, and average backfat and markedly decreased drip loss (p < 0.05) of finishing pigs compared with the control group. Microbial analysis of fecal samples revealed a marked increase in α-diversity and significantly altered composition of gut microbiota in finishing pigs in response to L-Citrulline. In particular, these altered gut microbiota at the phylum and genus level may be mainly involved in the metabolic process of carbohydrate, energy, and amino acid, and exhibited a significant association with final weight, carcass weight, and backfat thickness. Taken together, our data revealed the potential role of L-Citrulline in the modulation of growth performance, carcass characteristics, and the meat quality of finishing pigs, which is most likely associated with gut microbiota.

Characteristics of microRNAs in Skeletal Muscle of Intrauterine Growth-Restricted Pigs.

microRNAs are a class of small RNAs that have been extensively studied, which are involved in many biological processes and disease occurrence. The incidence of intrauterine growth restriction is higher in mammals, especially multiparous mammals. In this study, we found that the weight of the longissimus dorsi of intrauterine growth-restricted pigs was significantly lower than that of normal pigs. Then, intrauterine growth-restricted pig longissimus dorsi were used to characterize miRNA expression profiles by RNA sequencing. A total of 333 miRNAs were identified, of which 26 were differentially expressed. Functional enrichment analysis showed that these differentially expressed miRNAs regulate the expression of their target genes (such as PIK3R1, CCND2, AKT3, and MAP3K7), and these target genes play an important role in the proliferation and differentiation of skeletal muscle through signaling pathways such as the PI3K-Akt, MAPK, and FoxO signaling pathways. Furthermore, miRNA-451 was significantly upregulated in IUGR pig skeletal muscle. Overexpression of miR-451 in C2C12 cells significantly promoted the expression of Mb, Myod, Myog, Myh1, and Myh7, suggesting that miR-451 may be involved in the regulation of the myoblastic differentiation of C2C12 cells. Our results reveal the role of miRNA-451 in regulating myogenic differentiation of skeletal muscle in pigs with intrauterine growth restriction.

Effect of Slightly Acidic Electrolyzed Water on Growth, Diarrhea and Intestinal Bacteria of Newly Weaned Piglets.

As an environmentally-friendly agent, slightly acidic electrolyzed water (SAEW) was introduced in drinking water of newly weaned piglets for diarrhea prevention. In total, 72 piglets were employed and 3% SAEW was added into the normal temperature and warm (30 °C) tap water, respectively, for this 33-day feeding experiment. It was found that the total bacteria and coliforms in the drinking water were reduced by 70% and 100%, respectively, with the addition of 3% SAEW. After SAEW treatment, the average daily water and feed intakes of piglets were increased during the first 16 days, and the diarrhea rate was reduced by 100%, with not one case of diarrhea recorded at the end of the experiment. The microbiome results demonstrated that SAEW decreased the diversity of caecum bacteria with normal tap water supplied, and increased the richness of the caecum bacteria with warm tap water supplied. SAEW also increased the abundance of potentially beneficial genera Sutterella and Ruminococcaceae_UCG-005 and reduced the abundance of pathogenic Faecalibacterium. Moreover, twelve metabolic functions belonging to the cluster of metabolism and organismal functions, including digestion and the endocrine and excretory systems, were greatly enhanced. Correlation analysis indicated that the influence of intestinal pathogens on water and feed intakes and the diarrhea of piglets were decreased by SAEW. The results suggest that SAEW can be used as an antibiotic substitute to prevent diarrhea in newly weaned piglets.

Estimation of genetic parameters of pig reproductive traits.

Introduction: In this study, we aimed to estimate the genetic parameters of the reproductive traits in three popular commercial pig breeds: Duroc, Landrace, and Yorkshire. Additionally, we evaluated the factors that influence these traits. Method: We collected data from a large number of litters, including 1,887 Duroc, 21,787 Landrace, and 74,796 Yorkshire litters. Using the ASReml-R software to analyze 11 traits, which included: total number of pigs born (TNB); number of piglets born alive (NBA); number of piglets born healthy (NBH); number of piglets born weak (NBW); number of new stillborn piglets (NS); number of old stillborn piglets (OS); number of piglets born with malformation (NBM); number of mummified piglets (NM); total litter birthweight (LBW); litter average weight (LAW); duration of gestational period (GP). We investigated the effects of 4 fixed factors on the genetic parameters of these traits. Results: Among the 11 reproductive-related traits, the gestational period belonged to the medium heritability traits (0.251-0.430), while remaining traits showed low heritability, ranging from 0.005 to 0.159. TNB, NBA, NBH, LBW had positive genetic correlation (0.737 ~ 0.981) and phenotype correlation (0.711 ~ 0.951). There was a negative genetic correlation between NBW and LAW (-0.452 ~ -0.978) and phenotypic correlation (-0.380 ~ -0.873). LBW was considered one of the most reasonable reproductive traits that could be used for breeding improvement. Repeatability of the three varieties was within the range of 0.000-0.097. In addition, the fixed effect selected in this study had a significant effect on Landrace and Yorkshire (p < 0.05). Discussion: We found a positive correlation between LBW and TNB, NBA, and NBH, suggesting the potential for multi-trait association breeding. Factors such as farm, farrowing year, breeding season, and parity should be taken into consideration in practical production, as they may impact the reproductive performance of breeding pigs.

Dynamic chromatin architecture of the porcine adipose tissues with weight gain and loss.

Using an adult female miniature pig model with diet-induced weight gain/weight loss, we investigated the regulatory mechanisms of three-dimensional (3D) genome architecture in adipose tissues (ATs) associated with obesity. We generated 249 high-resolution in situ Hi-C chromatin contact maps of subcutaneous AT and three visceral ATs, analyzing transcriptomic and chromatin architectural changes under different nutritional treatments. We find that chromatin architecture remodeling underpins transcriptomic divergence in ATs, potentially linked to metabolic risks in obesity development. Analysis of chromatin architecture among subcutaneous ATs of different mammals suggests the presence of transcriptional regulatory divergence that could explain phenotypic, physiological, and functional differences in ATs. Regulatory element conservation analysis in pigs and humans reveals similarities in the regulatory circuitry of genes responsible for the obesity phenotype and identified non-conserved elements in species-specific gene sets that underpin AT specialization. This work provides a data-rich tool for discovering obesity-related regulatory elements in humans and pigs.

Potential Function of Testicular MicroRNAs in Heat-Stress-Induced Spermatogenesis Disorders.

Spermatogenesis is temperature-dependent, and the increase in testicular temperature seriously affects mammalian spermatogenesis and semen quality. In this study, the testicular heat stress model of mice was made with a 43 °C water bath for 25 min, and the effects of heat stress on semen quality and spermatogenesis-related regulators were analyzed. On the 7th day after heat stress, testis weight shrank to 68.45% and sperm density dropped to 33.20%. High-throughput sequencing analysis showed that 98 microRNAs (miRNAs) and 369 mRNAs were down-regulated, while 77 miRNAs and 1424 mRNAs were up-regulated after heat stress. Through gene ontology (GO) analysis of differentially expressed genes and miRNA-mRNA co-expression networks, it was found that heat stress may be involved in the regulation of testicular atrophy and spermatogenesis disorders by affecting cell meiosis process and cell cycle. In addition, through functional enrichment analysis, co-expression regulatory network, correlation analysis and in vitro experiment, it was found that miR-143-3p may be a representative potential key regulatory factor affecting spermatogenesis under heat stress. In summary, our results enrich the understanding of miRNAs in testicular heat stress and provide a reference for the prevention and treatment of heat-stress-induced spermatogenesis disorders.

n-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n-3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n-3 PUFA addition and showed that n-3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n-3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n-3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n-3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n-3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.

tsRNA Landscape and Potential Function Network in Subcutaneous and Visceral Pig Adipose Tissue.

Noncoding RNAs (ncRNAs) called tsRNAs (tRNA-derived short RNAs) have the ability to regulate gene expression. The information on tsRNAs in fat tissue is, however, limited. By sequencing, identifying, and analyzing tsRNAs using pigs as animal models, this research reports for the first time the characteristics of tsRNAs in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). A total of 474 tsRNAs, 20 and 21 of which were particularly expressed in VAT and SAT, respectively, were found in WAT. According to the analysis of the tsRNA/miRNA/mRNA co-expression network, the tsRNAs with differential expression were primarily engaged in the endocrine and immune systems, which fall under the classification of organic systems, as well as the global and overview maps and lipid metropolis, which fall under the category of metabolism. This research also discovered a connection between the activity of the host tRNA engaged in translation and the production of tsRNAs. This research also discovered that tRF-Gly-GCC-037/tRF-Gly-GCC-042/tRF-Gly-CCC-016 and miR-218a/miR281b may be involved in the regulation of fatty acid metabolism in adipose tissue through SCD based on the tsRNA/miRNA/mRNA/fatty acid network. In conclusion, our findings enrich the understanding of ncRNAs in WAT metabolism and health regulation, as well as reveal the differences between SAT and VAT at the level of tsRNAs.

DNA Damage-Induced, S-Phase Specific Phosphorylation of Orc6 is Critical for the Maintenance of Genome Stability.

The smallest subunit of the human Origin Recognition Complex, hOrc6, is required for DNA replication progression and plays an important role in mismatch repair (MMR) during S-phase. However, the molecular details of how hOrc6 regulates DNA replication and DNA damage response remain to be elucidated. Orc6 levels are elevated upon specific types of genotoxic stress, and it is phosphorylated at Thr229, predominantly during S-phase, in response to oxidative stress. Many repair pathways, including MMR, mediate oxidative DNA damage repair. Defects in MMR are linked to Lynch syndrome, predisposing patients to many cancers, including colorectal cancer. Orc6 levels are known to be elevated in colorectal cancers. Interestingly, tumor cells show reduced hOrc6-Thr229 phosphorylation compared to adjacent normal mucosa. Further, elevated expression of wild-type and the phospho-dead forms of Orc6 results in increased tumorigenicity, implying that in the absence of this "checkpoint" signal, cells proliferate unabated. Based on these results, we propose that DNA-damage-induced hOrc6-pThr229 phosphorylation during S-phase facilitates ATR signaling in the S-phase, halts fork progression, and enables assembly of repair factors to mediate efficient repair to prevent tumorigenesis. Our study provides novel insights into how hOrc6 regulates genome stability.