The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames.

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.

A novel lncRNA-hidden polypeptide regulates malignant phenotypes and pemetrexed sensitivity in A549 pulmonary adenocarcinoma cells.

The advance of high-throughput sequencing enhances the discovery of short ORFs embedded in long non-coding RNAs (lncRNAs). Here, we uncovered the production and biological activity of lncRNA-hidden polypeptides in lung adenocarcinoma (LUAD). In the present study, bioinformatics was used to screen the lncRNA-hidden polypeptides in LUAD. Analysis of protein expression was done by western blot or immunofluorescence assay. The functions of the polypeptide were determined by detecting its effects on cell viability, proliferation, migration, invasion, and pemetrexed (PEM) sensitivity. The protein interactors of the polypeptide were analyzed by mass spectrometry after Co-immunoprecipitation (Co-IP) assay. The results showed that the lncRNA LINC00954 was confirmed to encode a novel polypeptide LINC00954-ORF. The polypeptide had tumor-suppressor features in A549 cells by repressing cell growth, motility and invasion. Moreover, the polypeptide enhanced PEM sensitivity and suppressed growth in A549/PEM cells. The protein interactors of this polypeptide had close correlations with RNA processing, amide metabolic process, translation, RNA binding, RNA transport, and DNA replication. As a conclusion, the LINC00954-ORF polypeptide embedded in lncRNA LINC00954 possesses tumor-suppressor features in A549 and PEM-resistant A549 cells and sensitizes PEM-resistant A549 cells to PEM, providing evidence that the LINC00954-ORF polypeptide is a potential anti-cancer agent in LUAD.

Small Proteins in Archaea, a Mainly Unexplored World.

In recent years, increasing numbers of small proteins have moved into the focus of science. Small proteins have been identified and characterized in all three domains of life, but the majority remains functionally uncharacterized, lack secondary structure, and exhibit limited evolutionary conservation. While quite a few have already been described for bacteria and eukaryotic organisms, the amount of known and functionally analyzed archaeal small proteins is still very limited. In this review, we compile the current state of research, show strategies for systematic approaches for global identification of small archaeal proteins, and address selected functionally characterized examples. Besides, we document exemplarily for one archaeon the tool development and optimization to identify small proteins using genome-wide approaches.

Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria.

Genome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, beginning with a 5'-AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here, we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome-binding site of the 3'gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine-encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine-responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome-directed regulons. Regulon-level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes.

Evidence That Regulation of Pri-miRNA/miRNA Expression Is Not a General Rule of miPEPs Function in Humans.

Some miRNAs are located in RNA precursors (pri-miRNAs) annotated as long non-coding (lncRNAs) due to absence of long open reading frames (ORFs). However, recent studies have shown that some lnc pri-miRNAs encode peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants, three miPEPs have also been identified in humans. Herein, we found that a dozen human pri-miRNAs potentially encode miPEPs, as revealed by ribosome profiling and proteomic databases survey. So far, the only known function of plant miPEPs is to enhance the transcription of their own pri-miRNAs, thereby increasing the level and activity of their associated miRNAs and downregulating the expression of their target genes. To date, in humans, only miPEP133 was shown to promote a positive autoregulatory loop. We investigated whether other human miPEPs are also involved in regulating the expression of their miRNAs by studying miPEP155, encoded by the lnc MIR155HG, miPEP497, a sORF-encoded peptide within lnc MIR497HG, and miPEP200a, encoded by the pri-miRNA of miR-200a/miR-200b. We show that overexpression of these miPEPs is unable to impact the expression/activity of their own pri-miRNA/miRNAs in humans, indicating that the positive feedback regulation observed with plant miPEPs and human miPEP133 is not a general rule of human miPEP function.

Small Proteins Encoded by Unannotated ORFs are Rising Stars of the Proteome, Confirming Shortcomings in Genome Annotations and Current Vision of an mRNA.

Short ORF-encoded peptides and small proteins in eukaryotes have been hiding in the shadow of large proteins for a long time. Recently, improved identifications in MS-based proteomics and ribosome profiling resulted in the detection of large numbers of small proteins. The variety of functions of small proteins is also emerging. It seems to be the right time to reflect on why small proteins remained invisible. In addition to the obvious technical challenge of detecting small proteins, they were mostly forgotten from annotations and they escaped detection because they were not sought. In this review, we identify conventions that need to be revisited, including the assumption that mature mRNAs carry only one coding sequence. The large-scale discovery of small proteins and of their functions will require changing some paradigms and undertaking the annotation of ORFs that are still largely perceived as irrelevant coding information compared to already annotated coding sequences.

The Long History of the Diverse Roles of Short ORFs: sPEPs in Fungi.

Since the completion of the genome sequence of the model eukaryote Saccharomyces cerevisiae, there have been significant advancements in the field of genome annotation, in no small part due to the availability of datasets that make large-scale comparative analyses possible. As a result, since its completion there has been a significant change in annotated ORF size distribution in this first eukaryotic genome, especially in short ORFs (sORFs) predicted to encode polypeptides less than 150 amino acids in length. Due to their small size and the difficulties associated with their study, it is only relatively recently that these genomic features and the sORF-encoded peptides (sPEPs) they encode have become a focus of many researchers. Yet while this class of peptides may seem new and exciting, the study of this part of the proteome is nothing new in S. cerevisiae, a species where the biological importance of sPEPs has been elegantly illustrated over the past 30 years. Here the authors showcase a range of different sORFs found in S. cerevisiae and the diverse biological roles of their encoded sPEPs, and provide an insight into the sORFs found in other fungal species, particularly those pathogenic to humans.

Common and phylogenetically widespread coding for peptides by bacterial small RNAs.

BACKGROUND: While eukaryotic noncoding RNAs have recently received intense scrutiny, it is becoming clear that bacterial transcription is at least as pervasive. Bacterial small RNAs and antisense RNAs (sRNAs) are often assumed to be noncoding, due to their lack of long open reading frames (ORFs). However, there are numerous examples of sRNAs encoding for small proteins, whether or not they also have a regulatory role at the RNA level. METHODS: Here, we apply flexible machine learning techniques based on sequence features and comparative genomics to quantify the prevalence of sRNA ORFs under natural selection to maintain protein-coding function in 14 phylogenetically diverse bacteria. Importantly, we quantify uncertainty in our predictions, and follow up on them using mass spectrometry proteomics and comparison to datasets including ribosome profiling. RESULTS: A majority of annotated sRNAs have at least one ORF between 10 and 50 amino acids long, and we conservatively predict that 409±191.7 unannotated sRNA ORFs are under selection to maintain coding (mean estimate and 95% confidence interval), an average of 29 per species considered here. This implies that overall at least 10.3±0.5% of sRNAs have a coding ORF, and in some species around 20% do. 165±69 of these novel coding ORFs have some antisense overlap to annotated ORFs. As experimental validation, many of our predictions are translated in published ribosome profiling data and are identified via mass spectrometry shotgun proteomics. B. subtilis sRNAs with coding ORFs are enriched for high expression in biofilms and confluent growth, and S. pneumoniae sRNAs with coding ORFs are involved in virulence. sRNA coding ORFs are enriched for transmembrane domains and many are predicted novel components of type I toxin/antitoxin systems. CONCLUSIONS: We predict over two dozen new protein-coding genes per bacterial species, but crucially also quantified the uncertainty in this estimate. Our predictions for sRNA coding ORFs, along with predicted novel type I toxins and tools for sorting and visualizing genomic context, are freely available in a user-friendly format at http://disco-bac.web.pasteur.fr. We expect these easily-accessible predictions to be a valuable tool for the study not only of bacterial sRNAs and type I toxin-antitoxin systems, but also of bacterial genetics and genomics.

Identifying (non-)coding RNAs and small peptides: challenges and opportunities.

Over the past decade, high-throughput studies have identified many novel transcripts. While their existence is undisputed, their coding potential and functionality have remained controversial. Recent computational approaches guided by ribosome profiling have indicated that translation is far more pervasive than anticipated and takes place on many transcripts previously assumed to be non-coding. Some of these newly discovered translated transcripts encode short, functional proteins that had been missed in prior screens. Other transcripts are translated, but it might be the process of translation rather than the resulting peptides that serves a function. Here, we review annotation studies in zebrafish to discuss the challenges of placing RNAs onto the continuum that ranges from functional protein-encoding mRNAs to potentially non-functional peptide-producing RNAs to non-coding RNAs. As highlighted by the discovery of the novel signaling peptide Apela/ELABELA/Toddler, accurate annotations can give rise to exciting opportunities to identify the functions of previously uncharacterized transcripts.

The dark proteome: translation from noncanonical open reading frames.

Omics-based technologies have revolutionized our understanding of the coding potential of the genome. In particular, these studies revealed widespread unannotated open reading frames (ORFs) throughout genomes and that these regions have the potential to encode novel functional (micro-)proteins and/or hold regulatory roles. However, despite their genomic prevalence, relatively few of these noncanonical ORFs have been functionally characterized, likely in part due to their under-recognition by the broader scientific community. The few that have been investigated in detail have demonstrated their essentiality in critical and divergent biological processes. As such, here we aim to discuss recent advances in understanding the diversity of noncanonical ORFs and their roles, as well as detail biologically important examples within the context of the mammalian genome.

Function annotation of peptides generated from the non-coding regions of D. melanogaster genome.

De novo emergence of genes is the most fundamental form of genetic diversity that is attracting the attention of the scientific community. Identification of short open reading frames (sORFs) from the non-coding regions of different genomes has been leading this thought recently. The coding potential of these newly identified sORFs have been investigated through experimental and computational approaches in recent studies. In the present work we have tried to make peptides from intergenic sequences of D. melanogaster genome leading to therapeutic applications. Towards this goal of making novel peptides from non-coding genome, we have found strong computational evidence of 145 peptides with conformational stability from the intergenic sequences of D. melanogaster. The structure of these completely unique peptides was predicted using ab initio method. The function annotation of these peptides was carried out using this structural information. The newly generated proteins were categorised as DNA/Protein/ion binding proteins, electron transporters and a very few as enzymes too. Experimental studies can certainly provide validations to these preliminary findings. This work provides further evidence of untapped potential of non-coding genome.