High Polymorphism Levels of De Novo ORFs in a Yoruba Human Population.

During evolution, new open reading frames (ORFs) with the potential to give rise to novel proteins continuously emerge. A recent compilation of noncanonical ORFs with translation signatures in humans has identified thousands of cases with a putative de novo origin. However, it is not known which is their distribution in the population. Are they universally translated? Here, we use ribosome profiling data from 65 lymphoblastoid cell lines from individuals of Yoruba origin to investigate this question. We identify 2,587 de novo ORFs translated in at least one of the cell lines. In line with their de novo origin, the encoded proteins tend to be smaller than 100 amino acids and encode positively charged proteins. We observe that the de novo ORFs are more polymorphic in the population than the set of canonical proteins, with a substantial fraction of them being translated in only some of the cell lines. Remarkably, this difference remains significant after controlling for differences in the translation levels. These results suggest that variations in the level translation of de novo ORFs could be a relevant source of intraspecies phenotypic diversity in humans.

Evolutionary Trajectories of New Duplicated and Putative De Novo Genes.

The formation of new genes during evolution is an important motor of functional innovation, but the rate at which new genes originate and the likelihood that they persist over longer evolutionary periods are still poorly understood questions. Two important mechanisms by which new genes arise are gene duplication and de novo formation from a previously noncoding sequence. Does the mechanism of formation influence the evolutionary trajectories of the genes? Proteins arisen by gene duplication retain the sequence and structural properties of the parental protein, and thus they may be relatively stable. Instead, de novo originated proteins are often species specific and thought to be more evolutionary labile. Despite these differences, here we show that both types of genes share a number of similarities, including low sequence constraints in their initial evolutionary phases, high turnover rates at the species level, and comparable persistence rates in deeper branchers, in both yeast and flies. In addition, we show that putative de novo proteins have an excess of substitutions between charged amino acids compared with the neutral expectation, which is reflected in the rapid loss of their initial highly basic character. The study supports high evolutionary dynamics of different kinds of new genes at the species level, in sharp contrast with the stability observed at later stages.

Native RNA sequencing in fission yeast reveals frequent alternative splicing isoforms.

The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies higher than 20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make about 80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model.

LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.

siRNA enrichment in Argonaute 2-depleted Blattella germanica.

BACKGROUND: RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation. METHODS: Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced. RESULTS: Depleting AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that obtained siRNAs mapped non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals. CONCLUSIONS/GENERAL SIGNIFICANCE: The siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies.

DNMT1 Promotes Genome Methylation and Early Embryo Development in Cockroaches.

The influence of DNA methylation on gene behavior and its consequent phenotypic effects appear to be very important, but the details are not well understood. Insects offer a diversity of DNA methylation modes, making them an excellent lineage for comparative analyses. However, functional studies have tended to focus on quite specialized holometabolan species, such as wasps, bees, beetles, and flies. Here, we have studied DNA methylation in the hemimetabolan insect Blattella germanica. In this cockroach, a gene involved in DNA methylation, DNA methyltransferase 1 (DNMT1), is expressed in early embryogenesis. In our experiments, RNAi of DNMT1 reduces DNA methylation and impairs blastoderm formation. Using reduced representation bisulfite sequencing and transcriptome analyses, we observed that methylated genes are associated with metabolism and are highly expressed, whereas unmethylated genes are related to signaling and show low expression. Moreover, methylated genes show greater expression change and less expression variability than unmethylated genes.

Chronic fructose intake does not induce liver steatosis and inflammation in female Sprague-Dawley rats, but causes hypertriglyceridemia related to decreased VLDL receptor expression.

PURPOSE: Sugar-sweetened beverage intake is a risk factor for insulin resistance, dyslipidemia, fatty liver, and steatohepatitis (NASH). Sub-chronic supplementation of liquid fructose, but not glucose, in female rats increases liver and plasma triglycerides without inflammation. We hypothesized that chronic supplementation of fructose would cause NASH and liver insulin resistance. METHODS: We supplemented female Sprague-Dawley rats with water or either fructose or glucose 10% w/v solutions under isocaloric conditions for 7 months. At the end, plasma analytes, insulin, and adiponectin were determined, as well as liver triglyceride content and the expression of key genes controlling inflammation, fatty acid synthesis and oxidation, endoplasmic reticulum stress, and plasma VLDL clearance, by biochemical and histological methods. RESULTS: Although sugar-supplemented rats increased their energy intake by 50-60%, we found no manifestation of liver steatosis, fibrosis or necrosis, unchanged plasma or tissue markers of inflammation or fibrosis, and reduced liver expression of gluconeogenic enzymes, despite both sugars increased fatty acid synthesis, mTORC1, and IRE1 activity, while decreasing fatty acid oxidation and PPARα activity. Only fructose-supplemented rats were hypertriglyceridemic, showing a reduced expression of VLDL receptor and lipoprotein lipase in skeletal muscle and vWAT. Glucose-supplemented rats showed increased adiponectinemia, which would explain the different metabolic outcomes of the two sugars. CONCLUSIONS: Chronic liquid simple sugar supplementation, as the sole risk factor, is not enough for female rats to develop NASH and increased liver gluconeogenesis. Nevertheless, under isocaloric conditions, only fructose induced hypertriglyceridemia, thus confirming that also the type of nutrient matters in the development of metabolic diseases.

Chronic Liquid Fructose, but not Glucose, Supplementation Selectively Induces Visceral Adipose Tissue Leptin Resistance and Hypertrophy in Female Sprague-Dawley Rats.

SCOPE: The effect of chronic supplementation with simple-sugar solutions on leptin signaling in liver, hypothalamus, and visceral white adipose tissue (vWAT) is studied, which is designed to mimic the temporal pattern of consumption by humans. METHODS AND RESULTS: Solutions of fructose or glucose are isocalorically supplemented (7 months) in female Sprague-Dawley rats consuming ad libitum rodent chow. After sacrifice, plasma and tissue samples (liver, hypothalamus, and vWAT) are collected. Zoometric parameters, plasma analytes, and the tissue expression and activity of markers of leptin signaling are determined by biochemical and molecular biological methods. The two sugars cause different types of adiposopathy. Both sugars induce increases in plasma nonesterified fatty acids, and leptin resistance in the liver and the hypothalamus. Only fructose-supplemented rats show hyperleptinemia, and increased body weight due to a hypertrophy of vWAT, with no signs of leptin-mediated lipolysis. Glucose-supplemented rats show no significant changes in these parameters but present elevated plasma adiponectin concentrations, lipolysis, and inflammatory markers in vWAT, indicating a shift to a nonexpandable adipose tissue phenotype. CONCLUSION: Chronic consumption of fructose places a greater burden on metabolic homeostasis than equivalent consumption of glucose, inducing hyperleptinemia, generalized leptin resistance, and increased body weight due to expanded, hypertrophic vWAT.

The Addition of Liquid Fructose to a Western-Type Diet in LDL-R-/- Mice Induces Liver Inflammation and Fibrogenesis Markers without Disrupting Insulin Receptor Signalling after an Insulin Challenge.

A high consumption of fat and simple sugars, especially fructose, has been related to the development of insulin resistance, but the mechanisms involved in the effects of these nutrients are not fully understood. This study investigates the effects of a Western-type diet and liquid fructose supplementation, alone and combined, on insulin signalling and inflammation in low-density lipoprotein (LDL) receptor-deficient mice (LDL-R-/-). LDL-R-/- mice were fed chow or Western diet ±15% fructose solution for 12 weeks. Plasma glucose and insulin, and the expression of genes related to inflammation in the liver and visceral white adipose tissue (vWAT), were analysed. V-akt murine thymoma viral oncogene homolog-2 (Akt) activation was measured in the liver of the mice after a single injection of saline or insulin. None of the dietary interventions caused inflammation in vWAT, whereas the Western diet induced hepatic inflammation, which was further enhanced by liquid fructose, leading also to a significant increase in fibrogenesis markers. However, there was no change in plasma glucose or insulin, or insulin-induced Akt phosphorylation. In conclusion, hepatic inflammation and fibrogenesis markers induced by a Western diet supplemented with liquid fructose in LDL-R-/- mice are not associated with a significant impairment of hepatic insulin signalling.