Biosurfer for systematic tracking of regulatory mechanisms leading to protein isoform diversity.

Long-read RNA sequencing has shed light on transcriptomic complexity, but questions remain about the functionality of downstream protein products. We introduce Biosurfer, a computational approach for comparing protein isoforms, while systematically tracking the transcriptional, splicing, and translational variations that underlie differences in the sequences of the protein products. Using Biosurfer, we analyzed the differences in 32,799 pairs of GENCODE annotated protein isoforms, finding a majority (70%) of variable N-termini are due to the alternative transcription start sites, while only 9% arise from 5' UTR alternative splicing. Biosurfer's detailed tracking of nucleotide-to-residue relationships helped reveal an uncommonly tracked source of single amino acid residue changes arising from the codon splits at junctions. For 17% of internal sequence changes, such split codon patterns lead to single residue differences, termed "ragged codons". Of variable C-termini, 72% involve splice- or intron retention-induced reading frameshifts. We found an unusual pattern of reading frame changes, in which the first frameshift is closely followed by a distinct second frameshift that restores the original frame, which we term a "snapback" frameshift. We analyzed long read RNA-seq-predicted proteome of a human cell line and found similar trends as compared to our GENCODE analysis, with the exception of a higher proportion of isoforms predicted to undergo nonsense-mediated decay. Biosurfer's comprehensive characterization of long-read RNA-seq datasets should accelerate insights of the functional role of protein isoforms, providing mechanistic explanation of the origins of the proteomic diversity driven by the alternative splicing. Biosurfer is available as a Python package at https://github.com/sheynkman-lab/biosurfer.

An inner mitochondrial membrane microprotein from the SLC35A4 upstream ORF regulates cellular metabolism.

Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing translation under stress conditions. Computational and empirical methods have identified uORFs in the 5'-UTRs of approximately half of all mouse and human transcripts, making uORFs one the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs are under studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functionalmicroprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.

Long Intergenic Non-Coding RNAs of Human Chromosome 18: Focus on Cancers.

Malignant neoplasms are characterized by high molecular heterogeneity due to multilevel deregulation of gene expression and cellular functions. It is known that non-coding RNAs, including long intergenic non-coding RNAs (lincRNAs), can play significant roles in cancer biology. The current review focuses on a systematical analysis of genomic, transcriptomic, epigenomic, interactomic, and literature data on 65 lincRNAs of human chromosome 18 in the context of pan-cancer studies. The entire group of lincRNAs can be conditionally divided into 4 subgroups depending on experimental evidence on direct or indirect involvement in cancers and the biological associations with cancers, which we found during the data-mining process: the most studied (5 lincRNAs), moderately or poorly studied (11 lincRNAs), and understudied (31 lincRNAs). For the remaining 18 lincRNAs, data for analysis were fragmentary or missing. Among the key findings were the following: Of the lincRNAs of human chromosome 18, 40% have tissue-specific expression patterns, 22% of lincRNAs are known to have gene fusions, 40% of lincRNAs are prone to gene amplifications and/or deletions in cancers at a frequency greater than 3%, and 23% of lincRNAs are differentially expressed across cancer types, whereas 7% have subtype-specific expression patterns. LincRNAs' interactomes consist of 'master' microRNAs and 47 proteins (including cancer-associated proteins and microRNAs) that can interact with 3 or more lincRNAs. Functional enrichment analysis of a set of highly co-expressed genes retrieved for 17 lincRNAs in different cancer types indicated the potential associations of these lincRNAs with cellular signaling pathways. Six lincRNAs encoded small open-reading frame (smORF) proteins with emerging roles in cancers, and microRNAs as well as proteins with known functions in molecular carcinogenesis can bind to coding regions of smORFs. We identified seven transcriptomic signatures with potential prognostic value, consisting of two to seven different lincRNAs only. Taken together, the literature, biomedical, and molecular biology data analyzed indicated that only five of all lincRNAs of human chromosome 18 are cancer-associated, while eleven other lincRNAs have the tendency to be associated with cancers.

Compositional features analysis by machine learning in genome represents linear adaptation of monkeypox virus.

Introduction: The global headlines have been dominated by the sudden and widespread outbreak of monkeypox, a rare and endemic zoonotic disease caused by the monkeypox virus (MPXV). Genomic composition based machine learning (ML) methods have recently shown promise in identifying host adaptability and evolutionary patterns of virus. Our study aimed to analyze the genomic characteristics and evolutionary patterns of MPXV using ML methods. Methods: The open reading frame (ORF) regions of full-length MPXV genomes were filtered and 165 ORFs were selected as clusters with the highest homology. Unsupervised machine learning methods of t-distributed stochastic neighbor embedding (t-SNE), Principal Component Analysis (PCA), and hierarchical clustering were performed to observe the DCR characteristics of the selected ORF clusters. Results: The results showed that MPXV sequences post-2022 showed an obvious linear adaptive evolution, indicating that it has become more adapted to the human host after accumulating mutations. For further accurate analysis, the ORF regions with larger variations were filtered out based on the ranking of homology difference to narrow down the key ORF clusters, which drew the same conclusion of linear adaptability. Then key differential protein structures were predicted by AlphaFold 2, which meant that difference in main domains might be one of the internal reasons for linear adaptive evolution. Discussion: Understanding the process of linear adaptation is critical in the constant evolutionary struggle between viruses and their hosts, playing a significant role in crafting effective measures to tackle viral diseases. Therefore, the present study provides valuable insights into the evolutionary patterns of the MPXV in 2022 from the perspective of genomic composition characteristics analysis through ML methods.

Amino acid pattern reveals multi-functionality of ORF3 protein from HEV.

The smallest open reading frame (ORF) encoded protein ORF3 of hepatitis E virus (HEV), recently, has been demonstrated to perform multiple functions besides accessory roles. ORF3 could act as a target for vaccine against HEV infections. The IDR (intrinsically disordered region); IDP (ID protein)/IDPR (ID protein region), plays critical role in various regulatory functions of viruses. The dark proteome of HEV-ORF3 protein including its structure and function was systematically examined by computer predictors to explicate its role in viral pathogenesis and drug resistance beyond its functions as accessory viral protein. Amino acid distribution showed ORF3 enrichment with disorder-promoting residues (Ala, Pro, Ser, Gly) while deficiency in order-promoting residues (Asn, Ile, Phe, Tyr and Trp). Initial investigation revealed ORF3 as IDP (entirely disordered protein) or IDPR (proteins consisting of IDRs with structured globular domains). Structural examination revealed preponderance of disordered regions interpreting ORF3 as moderately/highly disordered protein. Further disorder predictors categorized ORF3 as highly disordered protein/IDP. Identified sites and associated-crucial molecular functions revealed ORF3 involvement in diverse biological processes, substantiating them as targets of regulation. As ORF3 functions are yet to completely explored, thus, data on its disorderness could help in elucidating its disorder related functions.

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.

Humanin variant P3S is associated with longevity in APOE4 carriers and resists APOE4-induced brain pathology.

The APOE4 allele is recognized as a significant genetic risk factor to Alzheimer's disease (AD) and influences longevity. Nonetheless, some APOE4 carriers exhibit resistance to AD even in advanced age. Humanin, a mitochondrial-derived peptide comprising 24 amino acids, has variants linked to cognitive resilience and longevity. Our research uncovered a unique humanin variant, P3S, specifically enriched in centenarians with the APOE4 allele. Through in silico analyses and subsequent experimental validation, we demonstrated a strong affinity between humanin P3S and APOE4. Utilizing an APOE4-centric mouse model of amyloidosis (APP/PS1/APOE4), we observed that humanin P3S significantly attenuated brain amyloid-beta accumulation compared to the wild-type humanin. Transcriptomic assessments of mice treated with humanin P3S highlighted its potential mechanism involving the enhancement of amyloid beta phagocytosis. Additionally, in vitro studies corroborated humanin P3S's efficacy in promoting amyloid-beta clearance. Notably, in the temporal cortex of APOE4 carriers, humanin expression is correlated with genes associated with phagocytosis. Our findings suggest a role of the rare humanin variant P3S, especially prevalent among individuals of Ashkenazi descent, in mitigating amyloid beta pathology and facilitating phagocytosis in APOE4-linked amyloidosis, underscoring its significance in longevity and cognitive health among APOE4 carriers.

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.

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, 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 over-oxidation in 2-oxoglutarate-dependent dioxygenases and iron mobilisation during seed development and germination.

[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.

Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.

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.

Taxonomic classification of genus Aeromonas using open reading frame-based binarized structure network analysis.

Objectives: Taxonomic assignment based on whole-genome sequencing data facilitates clear demarcation of species within a complex genus. Here, we applied a unique pan-genome phylogenetic method, open reading frame (ORF)-based binarized structure network analysis (OSNA), for taxonomic inference of Aeromonas spp., a complex taxonomic group consisting of 30 species. Methods: Data from 335 publicly available Aeromonas genomes, including the reference genomes of 30 species, were used to build a phylogenetic tree using OSNA. In OSNA, whole-genome structures are expressed as binary sequences based on the presence or absence of ORFs, and a tree is generated using neighbor-net, a distance-based method for constructing phylogenetic networks from binary sequences. The tree built by OSNA was compared to that constructed by a core-genome single-nucleotide polymorphism (SNP)-based analysis. Furthermore, the orthologous average nucleotide identity (OrthoANI) values of the sequences that clustered in a single clade in the OSNA-based tree were calculated. Results: The phylogenetic tree constructed with OSNA successfully delineated the majority of species of the genus Aeromonas forming conspecific clades for individual species, which was corroborated by OrthoANI values. Moreover, the OSNA-based phylogenetic tree demonstrated high compositional similarity to the core-genome SNP-based phylogenetic tree, supported by the Fowlkes-Mallows index. Conclusions: We propose that OSNA is a useful tool in predicting the taxonomic classification of complex bacterial genera.

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.

Combining a prioritization strategy and functional studies nominates 5'UTR variants underlying inherited retinal disease.

BACKGROUND: 5' untranslated regions (5'UTRs) are essential modulators of protein translation. Predicting the impact of 5'UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5'UTRs by different whole exome sequencing (WES) kits. The selected 5'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5'UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5'UTR compared to that of their canonical isoform. Depending on the probe design, 3-20% of IRD genes have 5'UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5'UTR variants implicated in IRDs and provides a systematic approach for 5'UTR annotation and validation that is applicable to other inherited diseases.

A descriptive study on spatial and temporal distributions of genetic clusters of porcine reproductive and respiratory syndrome virus infecting pig sites in Quebec, Canada, between 2010 and 2019.

BACKGROUND: The wide diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains combined with incomplete heterologous cross-protection complicates the management of the disease at both the herd and the regional levels. The objectives of this study were to describe the spatial and temporal distribution of various PRRSV genetic clusters infecting pig sites in Quebec, Canada, and to compare PRRSV regional diversity of wild-type sequences over the years. MATERIALS AND METHODS: A retrospective surveillance-based study was conducted on all pig sites which had PRRSV ORF5 sequences from field submissions transferred into the Laboratoire d'épidémiologie et de médecine porcine database from January 1, 2010 to December 31, 2019. A maximum likelihood phylogenetic tree inferred from multiple sequence alignment was used to identify genetic clusters. For each wild-type cluster gathering ≥ 15 sequences, the number of pig sites in which the cluster was detected per administrative region and per year were displayed on bubble charts and the spatiotemporal distribution of pig sites was illustrated using pie chart maps. A molecular analysis of variance was performed to compare PRRSV wild-type sequence diversity according to the administrative region for each year. RESULTS: A total of 32 wild-type clusters gathering 1653 PRRSV2 sequences from 693 pig sites were described. Each cluster was detected on up to 132 pig sites and 7 administrative regions over the 10-year period. Annually, the mean (min-max) number of wild-type clusters detected in at least one pig site reached 24 (17-29). Some clusters remained localized on a few sites over time whereas others were widespread over the territory during a few or many years. For each year, regional differences were also observed in PRRSV diversity of wild-type sequences. CONCLUSIONS: The differences observed in both the spatiotemporal distributions of PRRSV clusters and in the regional diversity of wild-type sequences highlight the importance of ongoing provincial surveillance to improve collective PRRS management strategies.

Decoding the Heterogeneity and Specialized Function of Translation Machinery Through Ribosome Profiling in Yeast Mutants of Initiation Factors.

The nuanced heterogeneity and specialized functions of translation machinery are increasingly recognized as crucial for precise translational regulation. Here, high-throughput ribosomal profiling (ribo-seq) is used to analyze the specialized roles of eukaryotic initiation factors (eIFs) in the budding yeast. By examining changes in ribosomal distribution across the genome resulting from knockouts of eIF4A, eIF4B, eIF4G1, CAF20, or EAP1, or knockdowns of eIF1, eIF1A, eIF4E, or PAB1, two distinct initiation-factor groups, the "looping" and "scanning" groups are discerned, based on similarities in the ribosomal landscapes their perturbation induced. The study delves into the cis-regulatory sequence features of genes influenced predominantly by each group, revealing that genes more dependent on the looping-group factors generally have shorter transcripts and poly(A) tails. In contrast, genes more dependent on the scanning-group factors often possess upstream open reading frames and exhibit a higher GC content in their 5' untranslated regions. From the ribosomal RNA fragments identified in the ribo-seq data, ribosomal heterogeneity associated with perturbation of specific initiation factors is further identified, suggesting their potential roles in regulating ribosomal components. Collectively, the study illuminates the complexity of translational regulation driven by heterogeneity and specialized functions of translation machinery, presenting potential approaches for targeted gene translation manipulation.

A method for evaluating of RNA's coding potential using the interaction effects of open reading frames and high-energy scalograms.

The identification and function determination of long non-coding RNAs (lncRNAs) can help to better understand the transcriptional regulation in both normal development and disease pathology, thereby demanding methods to distinguish them from protein-coding (pcRNAs) after obtaining sequencing data. Many algorithms based on the statistical, structural, physical, and chemical properties of the sequences have been developed for evaluating the coding potential of RNA to distinguish them. In order to design common features that do not rely on hyperparameter tuning and optimization and are evaluated accurately, we designed a series of features from the effects of open reading frames (ORFs) on their mutual interactions and with the electrical intensity of sequence sites to further improve the screening accuracy. Finally, the single model constructed from our designed features meets the strong classifier criteria, where the accuracy is between 82% and 89%, and the prediction accuracy of the model constructed after combining the auxiliary features equal to or exceed some best classification tools. Moreover, our method does not require special hyper-parameter tuning operations and is species insensitive compared to other methods, which means this method can be easily applied to a wide range of species. Also, we find some correlations between the features, which provides some reference for follow-up studies.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

Micropeptides: origins, identification, and potential role in metabolism-related diseases. / å¾®è‚½ï¼šèµ·æºã€é‰´å®šåŠå ¶åœ¨ä»£è°¢ç›¸å ³ç–¾ç— ä¸­çš„ä½œç”¨.

With the development of modern sequencing techniques and bioinformatics, genomes that were once thought to be noncoding have been found to encode abundant functional micropeptides (miPs), a kind of small polypeptides. Although miPs are difficult to analyze and identify, a number of studies have begun to focus on them. More and more miPs have been revealed as essential for energy metabolism homeostasis, immune regulation, and tumor growth and development. Many reports have shown that miPs are especially essential for regulating glucose and lipid metabolism and regulating mitochondrial function. MiPs are also involved in the progression of related diseases. This paper reviews the sources and identification of miPs, as well as the functional significance of miPs for metabolism-related diseases, with the aim of revealing their potential clinical applications.