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.

Functional Segregation of Overlapping Genes in HIV.

Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.

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.

Large-scale evidence for logarithmic effects of word predictability on reading time.

During real-time language comprehension, our minds rapidly decode complex meanings from sequences of words. The difficulty of doing so is known to be related to words' contextual predictability, but what cognitive processes do these predictability effects reflect? In one view, predictability effects reflect facilitation due to anticipatory processing of words that are predictable from context. This view predicts a linear effect of predictability on processing demand. In another view, predictability effects reflect the costs of probabilistic inference over sentence interpretations. This view predicts either a logarithmic or a superlogarithmic effect of predictability on processing demand, depending on whether it assumes pressures toward a uniform distribution of information over time. The empirical record is currently mixed. Here, we revisit this question at scale: We analyze six reading datasets, estimate next-word probabilities with diverse statistical language models, and model reading times using recent advances in nonlinear regression. Results support a logarithmic effect of word predictability on processing difficulty, which favors probabilistic inference as a key component of human language processing.

Mitochondrial protein synthesis quality control.

Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research.

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.

A Massive Proteogenomic Screen Identifies Thousands of Novel Peptides From the Human "Dark" Proteome.

Although the human gene annotation has been continuously improved over the past 2 decades, numerous studies demonstrated the existence of a "dark proteome", consisting of proteins that were critical for biological processes but not included in widely used gene catalogs. The Genotype-Tissue Expression project generated more than 15,000 RNA-seq datasets from multiple tissues, which modeled 30 million transcripts in the human genome. To provide a resource of high-confidence novel proteins from the dark proteome, we screened 50,000 mass spectrometry runs from over 900 projects to identify proteins translated from the Genotype-Tissue Expression transcript model with proteomic support. We also integrated 3.8 million common genetic variants from the gnomAD database to improve peptide identification. As a result, we identified 170,529 novel peptides with proteomic evidence, of which 6048 passed the strictest standard we defined and were supported by PepQuery. We provided a user-friendly website (https://ncorf.genes.fun/) for researchers to check the evidence of novel peptides from their studies. The findings will improve our understanding of coding genes and facilitate genomic data interpretation in biomedical research.

Spatiotemporally distributed frontotemporal networks for sentence reading.

Reading a sentence entails integrating the meanings of individual words to infer more complex, higher-order meaning. This highly rapid and complex human behavior is known to engage the inferior frontal gyrus (IFG) and middle temporal gyrus (MTG) in the language-dominant hemisphere, yet whether there are distinct contributions of these regions to sentence reading is still unclear. To probe these neural spatiotemporal dynamics, we used direct intracranial recordings to measure neural activity while reading sentences, meaning-deficient Jabberwocky sentences, and lists of words or pseudowords. We isolated two functionally and spatiotemporally distinct frontotemporal networks, each sensitive to distinct aspects of word and sentence composition. The first distributed network engages the IFG and MTG, with IFG activity preceding MTG. Activity in this network ramps up over the duration of a sentence and is reduced or absent during Jabberwocky and word lists, implying its role in the derivation of sentence-level meaning. The second network engages the superior temporal gyrus and the IFG, with temporal responses leading those in frontal lobe, and shows greater activation for each word in a list than those in sentences, suggesting that sentential context enables greater efficiency in the lexical and/or phonological processing of individual words. These adjacent, yet spatiotemporally dissociable neural mechanisms for word- and sentence-level processes shed light on the richly layered semantic networks that enable us to fluently read. These results imply distributed, dynamic computation across the frontotemporal language network rather than a clear dichotomy between the contributions of frontal and temporal structures.

Sex and age differences in "theory of mind" across 57 countries using the English version of the "Reading the Mind in the Eyes" Test.

The "Reading the Mind in the Eyes" Test (Eyes Test) is a widely used assessment of "theory of mind." The NIMH Research Domain Criteria recommends it as one of two tests for "understanding mental states." Previous studies have demonstrated an on-average female advantage on the Eyes Test. However, it is unknown whether this female advantage exists across the lifespan and across a large number of countries. Thus, we tested sex and age differences using the English version of the Eyes Test in adolescents and adults across 57 countries. We also tested for associations with sociodemographic and cognitive/personality factors. We leveraged one discovery dataset (N = 305,726) and three validation datasets (Ns = 642; 5,284; and 1,087). The results show that: i) there is a replicable on-average female advantage in performance on the Eyes Test; ii) performance increases through adolescence and shallowly declines across adulthood; iii) the on-average female advantage is evident across the lifespan; iv) there is a significant on-average female advantage in 36 out of 57 countries; v) there is a significant on-average female advantage on translated (non-English) versions of the Eyes Test in 12 out of 16 countries, as confirmed by a systematic review; vi) D-scores, or empathizing-systemizing, predict Eyes Test performance above and beyond sex differences; and vii) the female advantage is negatively linked to "prosperity" and "autonomy," and positively linked to "collectivism," as confirmed by exploratory country-level analyses. We conclude that the on-average female advantage on the Eyes Test is observed across ages and most countries.

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.

Emergence of two distinct spatial folds in a pair of plant virus proteins encoded by nested genes.

Virus genomes may encode overlapping or nested open reading frames that increase their coding capacity. It is not known whether the constraints on spatial structures of the two encoded proteins limit the evolvability of nested genes. We examine the evolution of a pair of proteins, p22 and p19, encoded by nested genes in plant viruses from the genus Tombusvirus. The known structure of p19, a suppressor of RNA silencing, belongs to the RAGNYA fold from the alpha+beta class. The structure of p22, the cell-to-cell movement protein from the 30K family widespread in plant viruses, is predicted with the AlphaFold approach, suggesting a single jelly-roll fold core from the all-beta class, structurally similar to capsid proteins from plant and animal viruses. The nucleotide and codon preferences impose modest constraints on the types of secondary structures encoded in the alternative reading frames, nonetheless allowing for compact, well-ordered folds from different structural classes in two similarly-sized nested proteins. Tombusvirus p22 emerged through radiation of the widespread 30K family, which evolved by duplication of a virus capsid protein early in the evolution of plant viruses, whereas lineage-specific p19 may have emerged by a stepwise increase in the length of the overprinted gene and incremental acquisition of functionally active secondary structure elements by the protein product. This evolution of p19 toward the RAGNYA fold represents one of the first documented examples of protein structure convergence in naturally occurring proteins.

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.

slORFfinder: a tool to detect open reading frames resulting from trans-splicing of spliced leader sequences.

Trans-splicing of a spliced leader (SL) to the 5' ends of mRNAs is used to produce mature mRNAs in several phyla of great importance to human health and the marine ecosystem. One of the consequences of the addition of SL sequences is the change or disruption of the open reading frames (ORFs) in the recipient transcripts. Given that most SL sequences have one or more of the trinucleotide NUG, including AUG in flatworms, trans-splicing of SL sequences can potentially supply a start codon to create new ORFs, which we refer to as slORFs, in the recipient mRNAs. Due to the lack of a tool to precisely detect them, slORFs were usually neglected in previous studies. In this work, we present the tool slORFfinder, which automatically links the SL sequences to the recipient mRNAs at the trans-splicing sites identified from SL-containing reads of RNA-Seq and predicts slORFs according to the distribution of ribosome-protected footprints (RPFs) on the trans-spliced transcripts. By applying this tool to the analyses of nematodes, ascidians and euglena, whose RPFs are publicly available, we find wide existence of slORFs in these taxa. Furthermore, we find that slORFs are generally translated at higher levels than the annotated ORFs in the genomes, suggesting they might have important functions. Overall, this study provides a tool, slORFfinder (https://github.com/songbo446/slORFfinder), to identify slORFs, which can enhance our understanding of ORFs in taxa with SL machinery.

Short open reading frame genes in innate immunity: from discovery to characterization.

Next-generation sequencing (NGS) technologies have greatly expanded the size of the known transcriptome. Many newly discovered transcripts are classified as long noncoding RNAs (lncRNAs) which are assumed to affect phenotype through sequence and structure and not via translated protein products despite the vast majority of them harboring short open reading frames (sORFs). Recent advances have demonstrated that the noncoding designation is incorrect in many cases and that sORF-encoded peptides (SEPs) translated from these transcripts are important contributors to diverse biological processes. Interest in SEPs is at an early stage and there is evidence for the existence of thousands of SEPs that are yet unstudied. We hope to pique interest in investigating this unexplored proteome by providing a discussion of SEP characterization generally and describing specific discoveries in innate immunity.

ORF3c is expressed in SARS-CoV-2-infected cells and inhibits innate sensing by targeting MAVS.

Most SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While the majority of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. One example is the ORF3a sgRNA that also encodes ORF3c, an enigmatic 41-amino-acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2-infected cells and suppresses RIG-I- and MDA5-mediated IFN-ß induction. ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage, and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c, demonstrating that this reading frame is not essential for efficient viral replication in vivo and is likely compensated by other viral proteins. In agreement with this, disruption of ORF3c does not significantly affect SARS-CoV-2 replication in CaCo-2, CaLu-3, or Rhinolophus alcyone cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.

Dissociation of reading and naming in ventral occipitotemporal cortex.

Lesions in the language-dominant ventral occipitotemporal cortex (vOTC) can result in selective impairment of either reading or naming, resulting in alexia or anomia. Yet, functional imaging studies that show differential activation for naming and reading do not reveal activity exclusively tuned to one of these inputs. To resolve this dissonance in the functional architecture of the vOTC, we used focused stimulation to the vOTC in 49 adult patients during reading and naming, and generated a population-level, probabilistic map to evaluate if reading and naming are clearly dissociable within individuals. Language mapping (50 Hz, 2829 stimulations) was performed during passage reading (216 positive sites) and visual naming (304 positive sites). Within the vOTC, we isolated sites that selectively disrupted reading (24 sites in 11 patients) or naming (27 sites in 12 patients), and those that disrupted both processes (75 sites in 21 patients). The anteromedial vOTC had a higher probability of producing naming disruption, while posterolateral regions resulted in greater reading-specific disruption. Between them lay a multi-modal region where stimulation disrupted both reading and naming. This work provides a comprehensive view of vOTC organization-the existence of a heteromodal cortex critical to both reading and naming, along with a causally dissociable unimodal naming cortex, and a reading-specific visual word form area in the vOTC. Their distinct roles as associative regions may thus relate to their connectivity within the broader language network that is disrupted by stimulation, more than to highly selective tuning properties. Our work also implies that pre-surgical mapping of both reading and naming is essential for patients requiring vOTC resections, as these functions are not co-localized, and such mapping may prevent the occurrence of unexpected deficits.

Subtitled speech: the neural mechanisms of ticker-tape synaesthesia.

The acquisition of reading modifies areas of the brain associated with vision and with language, in addition to their connections. These changes enable reciprocal translation between orthography and the sounds and meaning of words. Individual variability in the pre-existing cerebral substrate contributes to the range of eventual reading abilities, extending to atypical developmental patterns, including dyslexia and reading-related synaesthesias. The present study is devoted to the little-studied but highly informative ticker-tape synaesthesia, in which speech perception triggers the vivid and irrepressible perception of words in their written form in the mind's eye. We scanned a group of 17 synaesthetes and 17 matched controls with functional MRI, while they listened to spoken sentences, words, numbers or pseudowords (Experiment 1), viewed images and written words (Experiment 2) or were at rest (Experiment 3). First, we found direct correlates of the ticker-tape synaesthesia phenomenon: during speech perception, as ticker-tape synaesthesia was active, synaesthetes showed over-activation of left perisylvian regions supporting phonology and of the occipitotemporal visual word form area, where orthography is represented. Second, we provided support to the hypothesis that ticker-tape synaesthesia results from atypical relationships between spoken and written language processing: the ticker-tape synaesthesia-related regions overlap closely with cortices activated during reading, and the overlap of speech-related and reading-related areas is larger in synaesthetes than in controls. Furthermore, the regions over-activated in ticker-tape synaesthesia overlap with regions under-activated in dyslexia. Third, during the resting state (i.e. in the absence of current ticker-tape synaesthesia), synaesthetes showed increased functional connectivity between left prefrontal and bilateral occipital regions. This pattern might reflect a lowered threshold for conscious access to visual mental contents and might imply a non-specific predisposition to all synaesthesias with a visual content. These data provide a rich and coherent account of ticker-tape synaesthesia as a non-detrimental developmental condition created by the interaction of reading acquisition with an atypical cerebral substrate.