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Obesity's complex etiology due to the interplay of environment and genetics makes it a more challenging research and health problem. Some of the contributing genetic factors that have not yet been examined in detail entail mRNA polyadenylation (PA). Genes with multiple PA sites express mRNA isoforms differing in coding sequence or 3′UTR through alternative polyadenylation (APA). Alterations in PA have been associated with various diseases however, its contribution to obesity is not well-researched. Following an 11-week high-fat diet, the APA sites in the hypothalamus of two unique mouse models for polygenic obesity (Fat line) and healthy leanness (Lean line) were determined using whole transcriptome termini site sequencing (WTTS-seq). We found 17 genes of interest with differentially expressed APA (DE-APA) isoforms, among which seven were previously associated with obesity or obesity-related traits (Pdxdc1, Smyd3, Rpl14, Copg1, Pcna, Ric3, Stx3) but have not yet been studied in the context of APA. The remaining ten genes (Ccdc25, Dtd2, Gm14403, Hlf, Lyrm7, Mrpl3, Pisd-ps3, Sbsn, Slx1b, Spon1) represent novel candidates associated with obesity/adiposity due to variability brought about by differential usage of APA sites. Our results provide insights into the relationship between PA and the hypothalamus in the context of obesity, by being the first study of DE-APA sites and DE-APA isoforms in these mouse models. Future studies are needed further to explore the role of APA isoforms in polygenic obesity by expanding the scope of research to other metabolically important tissues (such as liver and adipose tissues) and investigating the potential for targeting PA as a therapeutic strategy for obesity management.

Nalaganje maščevja je kompleksna lastnost, na katero med drugim vplivajo številni okoljski dejavniki in genetske razlike med posamezniki. Za preučevanje genetskih dejavnikov so najbolj primerni mišji poligeni modeli. V našem postopku smo uporabili liniji, ki sta bili selekcionirani na visok (debela linija) ali nizek (vitka linija) odstotek telesnega maščevja. Prejšnje študije so nakazale, da je gen Pla2g4e eden od potencialnih kandidatnih genov, ki lahko razloži del fenotipske razlike med linijama. Zato smo si kot prvi cilj zadali analizo izražanja Pla2g4e v hipotalamusu med vitko in debelo linijo, ki sta bili krmljeni s krmama z različno vsebnostjo maščob. Dokazali smo statistično značilno povišano izražanje gena v hipotalamusu debele linije miši, ne glede na vrsto krme. Drugi cilj je bil okarakterizirati gen Pla2g4e s sodobnimi bioinformacijskimi metodami, predvsem z vidika identifikacije polimorfizmov med vitko in debelo linijo, ki bi bili lahko odgovorni za diferencialno izražanje. Pri debeli liniji smo našli polimorfizme, zaradi katerih se lahko izgubijo vezavne regije za miRNA in transkripcijski represor CTCF, kar predvidevamo, da lahko pri tej liniji vodi v povišano izražanje gena Pla2g4e in posledično povišano raven nalaganja telesne maščobe. Pridobljeni rezultati kažejo, kako pomembno je genetsko ozadje pri debelosti in predstavljajo osnovo za razvoj potencialnih novih biooznačevalcev za nalaganje maščobe. Fat accumulation is a complex trait influenced by many environmental factors and genetic differences between individuals. Mouse polygenic models are best suited to study genetic factors. In our experiment, we used two lines selected for high (fat line) or low (lean line) body fat percentage. Previous studies have suggested that the gene Pla2g4e is one of the potential candidate genes that may explain part of the phenotypic difference between the lines. Therefore, as a first aim, we set out to analyse the expression of Pla2g4e in the hypothalamus between the lean and fat lines that were fed diets containing different levels of fat. We demonstrated a statistically significant increased gene expression in the hypothalamus of the obese mouse line, irrespective of the diet type or sex. The second objective was to characterise the gene Pla2g4e using bioinformatics methods with the objective of identifying polymorphisms between the lean and the obese lines that might be responsible for the differential expression. In the obese line, we found polymorphisms that may result in a loss of binding regions for miRNA and the transcriptional repressor CTCF, which we hypothesise may lead to increased expression of the gene Pla2g4e and consequently increased levels of body fat deposition in this line. These results demonstrate the importance of genetic background in obesity and provide a basis for the development of potential new biomarkers for fat deposition.

Alternative polyadenylation (APA) determines mRNA stability, localisation, translation and protein function. Several diseases, including obesity, have been linked to APA. Studies have shown that single nucleotide polymorphisms in polyadenylation signals (PAS-SNPs) can influence APA and affect phenotype and disease susceptibility. However, these studies focussed on associations between single PAS-SNP alleles with very large effects and phenotype. Therefore, we performed a genome-wide screening for PAS-SNPs in the polygenic mouse selection lines for fatness and leanness by whole-genome sequencing. The genetic variants identified in the two lines were overlapped with locations of PAS sites obtained from the PolyASite 2.0 database. Expression data for selected genes were extracted from the microarray expression experiment performed on multiple tissue samples. In total, 682 PAS-SNPs were identified within 583 genes involved in various biological processes, including transport, protein modifications and degradation, cell adhesion and immune response. Moreover, 63 of the 583 orthologous genes in human have been previously associated with human diseases, such as nervous system and physical disorders, and immune, endocrine, and metabolic diseases. In both lines, PAS-SNPs have also been identified in genes broadly involved in APA, such as Polr2c, Eif3e and Ints11. Five PAS-SNPs within 5 genes (Car, Col4a1, Itga7, Lat, Nmnat1) were prioritised as potential functional variants and could contribute to the phenotypic disparity between the two selection lines. The developed PAS-SNPs catalogue presents a key resource for planning functional studies to uncover the role of PAS-SNPs in APA, disease susceptibility and fat deposition.