Large-Scale Analyses of Human Microbiomes Reveal Thousands of Small, Novel Genes.

Small proteins are traditionally overlooked due to computational and experimental difficulties in detecting them. To systematically identify small proteins, we carried out a comparative genomics study on 1,773 human-associated metagenomes from four different body sites. We describe >4,000 conserved protein families, the majority of which are novel; ∼30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain and almost half are not represented in reference genomes. We identify putative housekeeping, mammalian-specific, defense-related, and protein families that are likely to be horizontally transferred. We provide evidence of transcription and translation for a subset of these families. Our study suggests that small proteins are highly abundant and those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.

Rethinking Unconventional Translation in Neurodegeneration.

Eukaryotic translation is tightly regulated to ensure that protein production occurs at the right time and place. Recent studies on abnormal repeat proteins, especially in age-dependent neurodegenerative diseases caused by nucleotide repeat expansion, have highlighted or identified two forms of unconventional translation initiation: usage of AUG-like sites (near cognates) or repeat-associated non-AUG (RAN) translation. We discuss how repeat proteins may differ due to not just unconventional initiation, but also ribosomal frameshifting and/or imperfect repeat DNA replication, expansion, and repair, and we highlight how research on translation of repeats may uncover insights into the biology of translation and its contribution to disease.

Upstream open reading frames control PLK4 translation and centriole duplication in primordial germ cells.

Centrosomes are microtubule-organizing centers comprised of a pair of centrioles and the surrounding pericentriolar material. Abnormalities in centriole number are associated with cell division errors and can contribute to diseases such as cancer. Centriole duplication is limited to once per cell cycle and is controlled by the dosage-sensitive Polo-like kinase 4 (PLK4). Here, we show that PLK4 abundance is translationally controlled through conserved upstream open reading frames (uORFs) in the 5' UTR of the mRNA. Plk4 uORFs suppress Plk4 translation and prevent excess protein synthesis. Mice with homozygous knockout of Plk4 uORFs (Plk4 Δu/Δu ) are viable but display dramatically reduced fertility because of a significant depletion of primordial germ cells (PGCs). The remaining PGCs in Plk4 Δu/Δu mice contain extra centrioles and display evidence of increased mitotic errors. PGCs undergo hypertranscription and have substantially more Plk4 mRNA than somatic cells. Reducing Plk4 mRNA levels in mice lacking Plk4 uORFs restored PGC numbers and fully rescued fertility. Together, our data uncover a specific requirement for uORF-dependent control of PLK4 translation in counterbalancing the increased Plk4 transcription in PGCs. Thus, uORF-mediated translational suppression of PLK4 has a critical role in preventing centriole amplification and preserving the genomic integrity of future gametes.

Balancing the scales: fine-tuning Polo-like kinase 4 to ensure proper centriole duplication.

Polo-like kinase 4 (Plk4) is the master regulator of centriole assembly. Several evolutionarily conserved mechanisms strictly regulate Plk4 abundance and activity to ensure cells maintain a proper number of centrioles. In this issue of Genes & Development, Phan et al. (pp. 718-736) add to this growing list by describing a new mechanism of control that restricts Plk4 translation through competitive ribosome binding at upstream open reading frames (uORFs) in the mature Plk4 mRNA. Fascinatingly, this mechanism is especially critical in the development of primordial germ cells in mice that are transcriptionally hyperactive and thus exquisitely sensitive to Plk4 mRNA regulation.

The oncomicropeptide APPLE promotes hematopoietic malignancy by enhancing translation initiation.

Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.

Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway.

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1). Following activation, NF-κB dimers bind the MCPyV noncoding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB- and NTAR-dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

Translational landscape in human early neural fate determination.

Gene expression regulation in eukaryotes is a multi-level process, including transcription, mRNA translation and protein turnover. Many studies have reported sophisticated transcriptional regulation during neural development, but the global translational dynamics are still ambiguous. Here, we differentiate human embryonic stem cells (ESCs) into neural progenitor cells (NPCs) with high efficiency and perform ribosome sequencing and RNA sequencing on both ESCs and NPCs. Data analysis reveals that translational controls engage in many crucial pathways and contribute significantly to regulation of neural fate determination. Furthermore, we show that the sequence characteristics of the untranslated region (UTR) might regulate translation efficiency. Specifically, genes with short 5'UTR and intense Kozak sequence are associated with high translation efficiency in human ESCs, whereas genes with long 3'UTR are related to high translation efficiency in NPCs. In addition, we have identified four biasedly used codons (GAC, GAT, AGA and AGG) and dozens of short open reading frames during neural progenitor differentiation. Thus, our study reveals the translational landscape during early human neural differentiation and provides insights into the regulation of cell fate determination at the translational level.

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

Modulation of prion protein expression through cryptic splice site manipulation.

Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region. This exon is homologous to exon 2 in nonprimate species but contains a start codon that would yield an upstream open reading frame with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.

Impact of translational regulation on diel expression revealed by time-series ribosome profiling in Arabidopsis.

Plants have developed the ability to adjust to the day/night cycle through the expression of diel genes, which allow them to effectively respond to environmental changes and optimise their growth and development. Diel oscillations also have substantial implications in many physiological processes, including photosynthesis, floral development, and environmental stress responses. The expression of diel genes is regulated by a combination of the circadian clock and responses to environmental cues, such as light and temperature. A great deal of information is available on the transcriptional regulation of diel gene expression. However, the extent to which translational regulation is involved in controlling diel changes in expression is not yet clear. To investigate the impact of translational regulation on diel expression, we conducted Ribo-seq and RNA-seq analyses on a time-series sample of Arabidopsis shoots cultivated under a 12 h light/dark cycle. Our results showed that translational regulation is involved in about 71% of the genes exhibiting diel changes in mRNA abundance or translational activity, including clock genes, many of which are subject to both translational and transcriptional control. They also revealed that the diel expression of glycosylation and ion-transporter-related genes is mainly established through translational regulation. The expression of several diel genes likely subject to translational regulation through upstream open-reading frames was also determined.

Noncanonical microprotein regulation of immunity.

The immune system is highly regulated but, when dysregulated, suboptimal protective or overly robust immune responses can lead to immune-mediated disorders. The genetic and molecular mechanisms of immune regulation are incompletely understood, impeding the development of more precise diagnostics and therapeutics for immune-mediated disorders. Recently, thousands of previously unrecognized noncanonical microprotein genes encoded by small open reading frames have been identified. Many of these microproteins perform critical functions, often in a cell- and context-specific manner. Several microproteins are now known to regulate immunity; however, the vast majority are uncharacterized. Therefore, illuminating what is often referred to as the "dark proteome," may present opportunities to tune immune responses more precisely. Here, we review noncanonical microprotein biology, highlight recently discovered examples regulating immunity, and discuss the potential and challenges of modulating dysregulated immune responses by targeting microproteins.

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Mitochondrial-derived microprotein MOTS-c attenuates immobilization-induced skeletal muscle atrophy by suppressing lipid infiltration.

Mitochondrial open reading frame of the 12S ribosomal RNA type-c (MOTS-c), a mitochondrial microprotein, has been described as a novel regulator of glucose and lipid metabolism. In addition to its role as a metabolic regulator, MOTS-c prevents skeletal muscle atrophy in high fat-fed mice. Here, we examined the preventive effect of MOTS-c on skeletal muscle mass, using an immobilization-induced muscle atrophy model, and explored its underlying mechanisms. Male C57BL/6J mice (10 wk old) were randomly assigned to one of the three experimental groups: nonimmobilization control group (sterilized water injection), immobilization control group (sterilized water injection), and immobilization and MOTS-c-treated group (15 mg/kg/day MOTS-c injection). We used casting tape for the immobilization experiment. After 8 days of the experimental period, skeletal muscle samples were collected and used for Western blotting, RNA sequencing, and lipid and collagen assays. Immobilization reduced ∼15% of muscle mass, whereas MOTS-c treatment attenuated muscle loss, with only a 5% reduction. MOTS-c treatment also normalized phospho-AKT, phospho-FOXO1, and phospho-FOXO3a expression levels and reduced circulating inflammatory cytokines, such as interleukin-1b (IL-1ß), interleukin-6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1), in immobilized mice. Unbiased RNA sequencing and its downstream analyses demonstrated that MOTS-c modified adipogenesis-modulating gene expression within the peroxisome proliferator-activated receptor (PPAR) pathway. Supporting this observation, muscle fatty acid levels were lower in the MOTS-c-treated group than in the casted control mice. These results suggest that MOTS-c treatment inhibits skeletal muscle lipid infiltration by regulating adipogenesis-related genes and prevents immobilization-induced muscle atrophy.NEW & NOTEWORTHY MOTS-c, a mitochondrial microprotein, attenuates immobilization-induced skeletal muscle atrophy. MOTS-c treatment improves systemic inflammation and skeletal muscle AKT/FOXOs signaling pathways. Furthermore, unbiased RNA sequencing and subsequent assays revealed that MOTS-c prevents lipid infiltration in skeletal muscle. Since lipid accumulation is one of the common pathologies among other skeletal muscle atrophies induced by aging, obesity, cancer cachexia, and denervation, MOTS-c treatment could be effective in other muscle atrophy models as well.

Three-nucleotide periodicity of nucleotide diversity in a population enables the identification of open reading frames.

Accurate prediction of open reading frames (ORFs) is important for studying and using genome sequences. Ribosomes move along mRNA strands with a step of three nucleotides and datasets carrying this information can be used to predict ORFs. The ribosome-protected footprints (RPFs) feature a significant 3-nt periodicity on mRNAs and are powerful in predicting translating ORFs, including small ORFs (sORFs), but the application of RPFs is limited because they are too short to be accurately mapped in complex genomes. In this study, we found a significant 3-nt periodicity in the datasets of populational genomic variants in coding sequences, in which the nucleotide diversity increases every three nucleotides. We suggest that this feature can be used to predict ORFs and develop the Python package 'OrfPP', which recovers ~83% of the annotated ORFs in the tested genomes on average, independent of the population sizes and the complexity of the genomes. The novel ORFs, including sORFs, identified from single-nucleotide polymorphisms are supported by protein mass spectrometry evidence comparable to that of the annotated ORFs. The application of OrfPP to tetraploid cotton and hexaploid wheat genomes successfully identified 76.17% and 87.43% of the annotated ORFs in the genomes, respectively, as well as 4704 sORFs, including 1182 upstream and 2110 downstream ORFs in cotton and 5025 sORFs, including 232 upstream and 234 downstream ORFs in wheat. Overall, we propose an alternative and supplementary approach for ORF prediction that can extend the studies of sORFs to more complex genomes.

The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions.

Ascorbate (vitamin C) is one of the most abundant primary metabolites in plants. Its complex chemistry enables it to function as an antioxidant, as a free radical scavenger, and as a reductant for iron and copper. Ascorbate biosynthesis occurs via the mannose/l-galactose pathway in green plants, and the evidence for this pathway being the major route is reviewed. Ascorbate accumulation is leaves is responsive to light, reflecting various roles in photoprotection. GDP-l-galactose phosphorylase (GGP) is the first dedicated step in the pathway and is important in controlling ascorbate synthesis. Its expression is determined by a combination of transcription and translation. Translation is controlled by an upstream open reading frame (uORF) which blocks translation of the main GGP-coding sequence, possibly in an ascorbate-dependent manner. GGP associates with a PAS-LOV protein, inhibiting its activity, and dissociation is induced by blue light. While low ascorbate mutants are susceptible to oxidative stress, they grow nearly normally. In contrast, mutants lacking ascorbate do not grow unless rescued by supplementation. Further research should investigate possible basal functions of ascorbate in severely deficient plants involving prevention of iron overoxidation in 2-oxoglutarate-dependent dioxygenases and iron mobilization during seed development and germination.

Assessing the discriminability of PCR-based open reading frame typing versus single-nucleotide polymorphism analysis via draft whole-genome sequencing of methicillin-resistant Staphylococcus aureus in nosocomial transmission analysis.

Phylogenetic analysis based on single-nucleotide polymorphism (SNP)-based through whole-genome sequencing is recognized as the standard method for probing nosocomial transmission. However, the application of WGS is constrained by the high cost of equipment and the need for diverse analysis tools, which limits its widespread use in clinical laboratory settings. In Japan, the prevalent use of PCR-based open reading frame typing (POT) for tracing methicillin-resistant Staphylococcus aureus (MRSA) transmission routes is attributed to its simplicity and ease of use. Although POT's discriminatory power is considered insufficient for nosocomial transmission analysis, conclusive data supporting this notion is lacking. This study assessed the discriminatory capabilities of SNP analysis and POT across 64 clinical MRSA strains. All 21 MRSA strains of ST5/SCCmec IIa, having more than 16 SNPs, demonstrated distinct clones. Conversely, two strains shared the same POT number and were identified as group A. Among the 12 MRSA strains of ST8/SCCmec IVl with over nine SNPs, five fell into POT group B, and five into POT group C. All four MRSA strains of ST8/SCCmec IVa were classified into POT group D, although they included strains with more than 30 SNPs. Among the 27 MRSA strains of ST1/SCCmec IVa, 14 were classified into POT group E. However, except for two clusters (each comprising two or three strains), all had SNP counts >10 (Fig. 1-D). SNP analysis of MRSA in CC1/SCCmec IV showed that several strains had the same number of SNPs in POT number (106-183-37), even among bacteria with >100 SNPs, indicating POT's limited use in detailed nosocomial transmission analysis.

A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics.

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

SARS-CoV-2 ORF7a Mutation Found in BF.5 and BF.7 Sublineages Impacts Its Functions.

A feature of the SARS-CoV-2 Omicron subvariants BF.5 and BF.7 that recently circulated mainly in China and Japan was the high prevalence of the ORF7a: H47Y mutation, in which the 47th residue of ORF7a has been mutated from a histidine (H) to a tyrosine (Y). Here, we evaluated the effect of this mutation on the three main functions ascribed to the SARS-CoV-2 ORF7a protein. Our findings show that H47Y mutation impairs the ability of SARS-CoV-2 ORF7a to antagonize the type I interferon (IFN-I) response and to downregulate major histocompatibility complex I (MHC-I) cell surface levels, but had no effect in its anti-SERINC5 function. Overall, our results suggest that the H47Y mutation of ORF7a affects important functions of this protein, resulting in changes in virus pathogenesis.

[Research progress of non-coding RNA-encoding polypeptides in primary hepatocellular carcinoma].

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, with rapid progression and a poor prognosis. More and more studies have shown that there are small open reading frames (sORFs) on the molecular sequences of a large number of non-coding RNAs (ncRNAs), which can encode conserved peptides that play an important role in controlling the occurrence and development of HCC. This article introduces the discovery, prediction, and validation methods of ncRNA-encoding polypeptides and reviews its research progress, with the aim of providing new targets and ideas for early-stage diagnosis, targeted therapy, and prognosis assessment of HCC.