Accurate and complete gene annotations are indispensable for understanding how genome sequences encode biological functions. For more than twenty years, the GENCODE consortium has developed reference annotations for the human and mouse genomes, becoming a foundation for biomedical and genomics communities worldwide. Nevertheless, collections of important yet poorly-understood gene classes like long non-coding RNAs (lncRNAs) remain incomplete and scattered across multiple, uncoordinated catalogs. To address this, GENCODE has undertaken the most comprehensive lncRNA annotation effort to date. This is founded on the manually supervised computational annotation of full-length targeted long-read sequencing, on matched embryonic and adult tissues, of orthologous regions in human and mouse. Altogether 17,931 human genes (140,268 transcripts) and 22,784 mouse genes (136,169 transcripts) have been added to the GENCODE catalog representing a 2-fold and 6-fold growth in transcripts, respectively - the greatest increase in the number of annotated human genes since the sequencing of the human genome. Our targeted design assigned human-mouse orthologs at a rate beyond previous studies, tripling the number of human disease-associated lncRNAs that have mouse orthologs. Novel lncRNA genes consistently exhibit biological signals of functionality, and they greatly enhance the functional interpretability of the human genome. While poorly expressed in bulk RNA-Seq samples, many of them are highly expressed in specific cell populations, maybe even contributing to cell-type determination. The expanded GENCODE lncRNA annotations mark a critical step toward deciphering the human and mouse genomes.

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 >20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make ∼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.

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.

During evolution, new ORFs with the potential to give rise to novel proteins continuously emerge. A recent compilation of non-canonical 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 intra-species phenotypic diversity in humans.

Classical cancer germline-antigens (CGAs) are proteins that are expressed in the male germinal line but not in somatic tissues, and that can also become expressed in tumors. However, the vast majority of testis-specific transcripts are long non-coding RNAs (lncRNAs) rather than protein-coding genes. Since recent studies have shown that many lncRNAs contain non-canonical open reading frames (ncORFs) that are translated into small proteins, or microproteins, there could be a large class of non-canonical cancer-germline antigens (ncCGAs) that remains to be discovered. Here, we integrate ribosome profiling from human testis and cancer cell lines with paired tumor/normal transcriptomes from 917 patients across eight common cancer types to define a comprehensive catalog of ncCGAs. This set comprises 235 ncCGAs encoded by lncRNAs or mRNA untranslated regions (5'UTRs and 3'UTRs), compared to 192 canonical CGAs (cCGAs) with similar expression patterns. We show that ncCGAs are evolutionary young, consistent with recent de novo emergence in the rapidly evolving male germline. Moreover, a large fraction is expressed across multiple patients and cancer types, indicating recurrent reactivation mechanisms in tumors. We further find that ncCGAs are frequently located in cancer-amplified regions or associated with MYC or E2F-regulated pathways, which may explain their expression in cancer. Finally, we provide strong evidence that a subset of ncCGAs give rise to potentially immunogenic HLA class I bound peptides. Together, our results describe a previously unexplored class of tumor-restricted antigens with potential applications in cancer immunotherapy.

The analysis of ribosome profiling (Ribo-Seq) data has provided evidence that many eukaryotic mRNAs contain translated upstream or downstream ORFs (uORFs/dORFs), but the biological significance of this translation activity remains, for the most part, unknown. One of the principal limitations has been the lack of Ribo-Seq data from several closely related species, precluding the identification of cases in which translation is phylogenetically conserved. Here, by combining Ribo-Seq data from 100 different experiments, we identify 2,332 translated uORFs and 1,008 translated dORFs in S. cerevisiae , which result in microproteins that tend to be highly hydrophobic or positively charged. To study their phylogenetic conservation, we have generated Nanopore direct RNA sequencing data, together with Ribo-Seq data, from six additional Saccharomyces species, spanning an evolutionary period of around 16 million years. We have identified 195 translated S. cerevisiae uORFs that are also translated in other Saccharomyces species; these uORFs are translated at levels comparable to the main coding sequence and display signatures of purifying selection at the level of the encoded microproteins. In contrast, dORFs are translated at very low levels and they are rarely conserved, suggesting much more limited microprotein functionalization. We have also discovered that uORF translation is associated with the formation of alternative transcript isoforms encompassing the region containing the uORFs but not the main protein coding sequence, implying that some microproteins can be produced independently of the main protein product. This work significantly advances our understanding of how initially pervasive uORF translation can result in new microproteins, providing many new candidates for further functional studies.