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SUMMARY: Geraniol is one of the most abundant aromatic compounds in fresh tea leaves and contributes to the pleasant odor of tea products. Additionally, it functions as an airborne signal that interacts with other members of the ecosystem. To date, the regulation of the geraniol biosynthesis in tea plants remains to be investigated. In this study, a correlation test of the content of geraniol and its glycosides with gene expression data revealed that nudix hydrolase, CsNudix26, and its transcription factor, CsbHLH133 are involved in geraniol biosynthesis. In vitro enzyme assays and metabolic analyses of genetically modified tea plants confirmed that CsNudix26 is responsible for the formation of geraniol. Yeast one‐hybrid, dual‐luciferase reporter, and EMSA assays were used to verify the binding of CsbHLH133 to the CsNudix26 promoter. Overexpression of CsbHLH133 in tea leaves enhanced CsNudix26 expression and geraniol accumulation, whereas CsbHLH133 silencing reduced CsNudix26 transcript levels and geraniol content. Interestingly, CsbHLH133‐AS, produced by alternative splicing, was discovered and proved to be the primary transcript expressed in response to various environmental stresses. Furthermore, geraniol release was found to be affected by various factors that alter the expression patterns of CsbHLH133 and CsbHLH133‐AS. Our findings indicate that distinct transcript splicing patterns of CsbHLH133 regulate geraniol biosynthesis in tea plants in response to different regulatory factors. Significance Statement: Geraniol is one of the most abundant aromatic compounds in fresh tea leaves and contributes to the pleasant odor of tea products. The transcription factor CsbHLH133 regulates geraniol biosynthesis by activating the expression of CsNudix26 in tea plants. Our results delineate the transcriptional regulatory network involved in geraniol biosynthesis in Camellia sinensis. Alternative splicing serves as a mechanism for plant stress tolerance, secondary metabolism, and developmental plasticity. Changes in the transcription of CsbHLH133 through alternative splicing modulate its abundance and translation efficiency, enabling tea plants to adapt better to adverse environmental conditions. [ABSTRACT FROM AUTHOR]

Background: Iron (Fe) deficiency affects 30–50% of the world's population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5' leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF). Results: We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5' LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression. Conclusions: Together these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance. Highlight: We searched for translationally repressive uORFs within Fe homeostasis genes to find precision breeding targets for improved crop nutrition and discovered uORFs in IDEF1 and IDEF2. [ABSTRACT FROM AUTHOR]

The 5′ untranslated region (5′UTR) of many positive-stranded RNA viruses contain functional regulatory sequences. Here, we show that the porcine reproductive and respiratory syndrome virus (PRRSV), a member of arteriviruses, harbors small upstream open reading frames (uORFs) in its 5′UTR. Bioinformatics analysis shows that this feature is relatively well conserved among PRRSV strains and Arteriviridae. We also identified a uORF, namely uORF2, in the PRRSV strain JXwn06, that possesses translational activity and exerts a suppressive effect on the expression of the primary ORF evidenced by in vitro reporter assays. We tested its importance via reverse genetics by introducing a point mutation into the PRRSV infectious cDNA clone to inactivate the start codon of uORF2. The recovered mutant virus Mut2 surprisingly replicated to the same level as the wild-type virus (WT), but induced a higher level of inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) both in vitro and in animal experiments, correlating well with more severe lung injury and higher death rate. In line with this, over-expression of uORF2 in transfected cells significantly inhibited poly(I:C)-induced expression of inflammatory cytokines. Together, our data support the idea that uORF2 encodes a novel, functional regulator of PRRSV virulence despite of its short size. IMPORTANCE PRRSV has remained a major challenge to the world swine industry, but we still do not know much about its biology and pathogenesis. Here, we provide evidence to show that the 5′UTR of PRRSV strain JXwn06 harbors a functional uORF that has the coding capacity and regulates induction of inflammation as demonstrated by in vitro assays and animal experiment. The findings reveal a novel viral factor that regulates cellular inflammation and provide insight into the understanding of PRRSV pathogenesis. [ABSTRACT FROM AUTHOR]

Many previously unsuspected single-nucleotide polymorphisms (SNPs) can cause phenotypic variation by affecting transcript splicing and protein translation. Small RNAs are more challenging to study than protein-coding genes but are important contributors to phenotypic evolution. Structural variation (e.g., transposable element insertion, gene copy number variation, chromosomal inversion) is an extremely common source of phenotypic divergence. The sequence composition of supergenes that control complex multi-trait polymorphisms has been elucidated at an unprecedented pace by long-read sequencing. Taxon-specific genes, including non-coding RNAs, can have a profound impact on lineage-specific phenotypic diversification and adaptation. It has been well documented that mutations in coding DNA or cis -regulatory elements underlie natural phenotypic variation in many organisms. However, the development of sophisticated functional tools in recent years in a wide range of traditionally non-model systems have revealed many 'unusual suspects' in the molecular bases of phenotypic evolution, including upstream open reading frames (uORFs), cryptic splice sites, and small RNAs. Furthermore, large-scale genome sequencing, especially long-read sequencing, has identified a cornucopia of structural variation underlying phenotypic divergence and elucidated the composition of supergenes that control complex multi-trait polymorphisms. In this review article we highlight recent studies that demonstrate this great diversity of molecular mechanisms producing adaptive genetic variation and the panoply of evolutionary paths leading to the 'grandeur of life'. [ABSTRACT FROM AUTHOR]

Ascorbate is involved in numerous vital processes, in particular in response to abiotic but also biotic stresses whose frequency and amplitude increase with climate change. Ascorbate levels vary greatly depending on species, tissues, or stages of development, but also in response to stress. Since its discovery, the ascorbate biosynthetic pathway has been intensely studied and it appears that GDP- l -galactose phosphorylase (GGP) is the enzyme with the greatest role in the control of ascorbate biosynthesis. Like other enzymes of this pathway, its expression is induced by various environmental and also developmental factors. Although mRNAs encoding it are among the most abundant in the transcriptome, the protein is only present in very small quantities. In fact, GGP translation is repressed by a negative feedback mechanism involving a small open reading frame located upstream of the coding sequence (uORF). Moreover, its activity is inhibited by a PAS/LOV type photoreceptor, the action of which is counteracted by blue light. Consequently, this multi-level regulation of GGP would allow fine control of ascorbate synthesis. Indeed, experiments varying the expression of GGP have shown that it plays a central role in response to stress. This new understanding will be useful for developing varieties adapted to future environmental conditions. [ABSTRACT FROM AUTHOR]

This Special Issue was assembled to mark the 25th anniversary of the proposal of the d -mannose/ l -galactose (Smirnoff–Wheeler) ascorbate biosynthesis pathway in plants (Wheeler et al. , 1998). The issue aims to assess the current state of knowledge and to identify outstanding questions about ascorbate metabolism and functions in plants. [ABSTRACT FROM AUTHOR]

High-throughput ribosome profiling demonstrates the translation of thousands of small open reading frames located in the 5′ untranslated regions of messenger RNAs (upstream ORFs). Upstream ORF can both perform a regulatory function by influencing the translation of the downstream main ORF and encode a small functional protein or microprotein. In this work, we showed that the 5′ untranslated region of the PRPF19 mRNA encodes an upstream ORF that is translated in human cells. Inactivation of this upstream ORF reduces the viability of human cells. [ABSTRACT FROM AUTHOR]

Acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1) is an enzyme that promotes mitochondrial dysfunction by catalyzing pathological remodeling of cardiolipin. Upregulation of ALCAT1 protein expression by oxidative stress is implicated in the pathogenesis of age-related metabolic diseases, but the underlying molecular mechanisms remain elusive. In this study, we identified a highly conserved upstream open reading frame (uORF) at the 5′-untranslated region (5′-UTR) of ALCAT1 mRNA as a key regulator of ALCAT1 expression in response to oxidative stress. We show that the uORF serves as a decoy that prevents translation initiation of ALCAT1 under homeostatic condition. The inhibitory activity of the uORF on ALCAT1 mRNA translation is mitigated by oxidative stress but not ER stress, which requires the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Consequently, ablation of uORF or eIF2α phosphorylation at Ser51 renders ALCAT1 protein expression unresponsive to induction by oxidative stress. Taken together, our data show that the uORF links oxidative stress to translation control of ALCAT1 mRNAs through phosphorylation of eIF2α at Ser51. [Display omitted] • A conserved upstream open reading frame (uORF) is located at the 5′-untranslated region of ALCAT1 mRNA. • The uORF inhibits the translation initiation of ALCAT1 mRNA under homeostatic condition. • The inhibitory activity of the uORF on ALCAT1 mRNA translation is mitigated by oxidative stress, but not ER stress. • Phosphorylation of eIF2α at Ser51 is required for ALCAT1 mRNA translation by oxidative stress. [ABSTRACT FROM AUTHOR]

Internal ribosome entry site (IRES) is a special functional element in RNA, which can directly recruit ribosome to initiate protein translation independently of the 5' end cap structure. It has been found to be closely related to a variety of cell processes. Recently, IRES have attracted a lol of attention due to increasing evidence that they play an extremely important role in the regulation of circular RNA translation. This paper reviews the current mechanism of IRES-mediated translation regulation in eukaryotic cells, and summarizes the bioinformatics tools related to IRES elements. [ABSTRACT FROM AUTHOR]

Upstream open reading frames (uORFs) regulate gene expression by modulating (mostly repressing) main open reading frame (mORF) translation. The regulatory activity of uORFs is affected by their sequence features and varying environments. Polymorphisms at the translation level caused by uORF variations can underlie phenotypic diversity in plants. uORFs are selected during evolution in eukaryotes and might contribute to local adaptation, domestication, and improvement in plants. Evolutionary conservation analyses, development of Ribo-seq technologies, utilization of pan-genome or super-pangenome resources, improvement of bioinformatics tools and databases to predict functional uORFs and the effects of uORF variants, phenomics- and tranlatomics-based genetic mapping, and CRISPR-based uORF engineering will collectively accelerate the application of natural uORF variations in plant breeding. Taking advantage of natural variation promotes our understanding of phenotypic diversity and trait evolution, ultimately accelerating plant breeding, in which the identification of causal variations is critical. To date, sequence variations in the coding region and transcription level polymorphisms caused by variations in the promoter have been prioritized. An upstream open reading frame (uORF) in the 5′ untranslated region (5′ UTR) regulates gene expression at the post-transcription or translation level. In recent years, studies have demonstrated that natural uORF variations shape phenotypic diversity. This opinion article highlights recent researches and speculates on future directions for natural uORF variation in plants. [ABSTRACT FROM AUTHOR]

Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans- acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5′UTR and the coding sequences contain cis- acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans- acting elements that drive selective mRNA translation in response to environmental changes. [ABSTRACT FROM AUTHOR]

Thermospermine plays a critical role in negatively regulating xylem development in angiosperms. A mutant of Arabidopsis thaliana that is defective in thermospermine biosynthesis, acaulis5 (acl5), exhibits a dwarf phenotype with excessive xylem formation. Mechanistically thermospermine acts in attenuating the inhibitory effect of an evolutionarily conserved upstream open reading frame (uORF) on the main ORF of SAC51, which encodes a basic helix-loop-helix protein involved in xylem repression. Here, we revealed that a semidominant suppressor of acl5, sac503, which partially restores the acl5 phenotype, has a point mutation in the conserved uORF of SAC51 with no amino acid substitution in the deduced peptide sequence. In transgenic lines carrying the β-glucuronidase (GUS) reporter gene fused with the SAC51 5ʹ region containing the uORF, the mutant construct was shown to confer higher GUS activity than does the wild-type SAC51 construct. We confirmed that sac503 mRNA was more stable than SAC51 mRNA in acl5. These results suggest that the single-base change in sac503 positively affects the translation of its main ORF instead of thermospermine. We further found that the uORF-GUS fusion protein could be synthesized in planta from the wild-type and sac503 translational fusion constructs. [ABSTRACT FROM AUTHOR]

The complexities of translational strategies make this stage of implementing genetic information one of the most challenging to comprehend and, simultaneously, perhaps the most engaging. It is evident that this diverse range of strategies results not only from a long evolutionary history, but is also of paramount importance for refining gene expression and metabolic modulation. This notion is particularly accurate for organisms that predominantly exhibit biochemical and physiological reactions with a lack of behavioural ones. Plants are a group of organisms that exhibit such features. Addressing unfavourable environmental conditions plays a pivotal role in plant physiology. This is particularly evident with the changing conditions of global warming and the irrevocable loss or depletion of natural ecosystems. In conceptual terms, the plant response to abiotic stress comprises a set of elaborate and intricate strategies. This is influenced by a range of abiotic factors that cause stressful conditions, and molecular genetic mechanisms that fine-tune metabolic pathways allowing the plant organism to overcome non-standard and non-optimal conditions. This review aims to focus on the current state of the art in the field of translational regulation in plants under abiotic stress conditions. Different regulatory elements and patterns are being assessed chronologically. We deem it important to focus on significant high-performance techniques for studying the genetic information dynamics during the translation phase. [ABSTRACT FROM AUTHOR]

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep. [ABSTRACT FROM AUTHOR]

uORF (upstream open reading frame) is a kind of mRNA element that can accurately control protein translation. It is located in the 5' leader region of mRNA. It has been reported to regulate the translation rate of downstream mORF (main open reading frame) by inhibiting the initiation of translation. Currently, the prediction and identification of plant uORF mainly focus on bioinformatics prediction and translatomics identification technology. uORF has regulatory roles on plants in many aspects, such as growth and development, nutrition and metabolism, disease resistance and immunity. In this review, we summarize the classification, functional mechanism, prediction and identification methods of plant uORF, strategies for plants to evade uORF, and the application progress of plant uORF in engineering. The purpose of this paper is to gain a more systematic and in-depth understanding of the function and mechanism of plant uORFs, and to provide a reference for the application of uORF in crop molecular breeding. [ABSTRACT FROM AUTHOR]

As it is well known, messenger RNA has many regulatory regions along its sequence length. One of them is the 5′ untranslated region (5'UTR), which itself contains many regulatory elements such as upstream ORFs (uORFs), internal ribosome entry sites (IRESs), microRNA binding sites, and structural components involved in the regulation of mRNA stability, pre-mRNA splicing, and translation initiation. Activation of the alternative, more upstream transcription start site leads to an extension of 5′UTR. One of the consequences of 5′UTRs extension may be head-to-head gene overlap. This review describes elements in 5′UTR of protein-coding transcripts and the functional significance of protein-coding genes 5′ overlap with implications for transcription, translation, and disease. [ABSTRACT FROM AUTHOR]

Many eukaryotic transcripts contain upstream open reading frames (uORFs). Translated uORFs can inhibit the translation of main ORFs by imposing the need for reinitiation of translation. Translated uORFs can also lead to transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. In mammalian cells, translated uORFs were shown to target their transcripts to NMD if the uORFs were long (>23–32 amino acids), structured, or inhibit reinitiation. Reinitiation was shown to rescue uORF-containing mammalian transcripts from NMD. Much less is known about the significance of the length, structure, and reinitiation efficiency of translated uORFs for NMD targeting in plants. Although high-throughput studies suggested that uORFs do not globally reduce plant transcript abundance, it was not clear whether this was due to NMD-escape-permitting parameters of uORF recognition, length, structure, or reinitiation efficiency. We expressed in Arabidopsis reporter genes that included NDL2 5ʹ untranslated region and various uORFs with modulation of the above parameters. We found that transcripts can escape NMD in plants even when they include efficiently translated uORFs up to 70 amino acids long, or structured uORFs, in the absence of reinitiation. These data highlight an apparent difference between the rules that govern the exposure of uORF-containing transcripts to NMD in mammalian and plant cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • TCP-seq delineates leaky scanning/reinitiation dichotomy in uORF-mediated translation control. • Novel insights into uORF regulation revealed through qualitative analysis of TCP-Seq profiles. • Selective TCP-seq profiles elucidate roles of specific initiation factors in reinitiation at individual uORFs. Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5′ leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including EIF5 , IFRD1 , MDM2 , MIEF1 , PPP1R15B , TAF7, and UCP2. [ABSTRACT FROM AUTHOR]

Proteins that bind to each mRNA may affect the latter's abundance and location in the cell and how well ribosomes will translate that mRNA into a protein. Hence, mRNA-binding proteins (mRBPs) represent obvious control points in gene expression. Surprisingly, little is known about mRBPs and cell-cycle progression. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 infection generates up to nine different sub-genomic mRNAs (sgRNAs), in addition to the genomic RNA (gRNA). The 5′UTR of each viral mRNA shares the first 75 nucleotides (nt.) at their 5′end, called the leader, but differentiates by a variable sequence (0 to 190 nt. long) that follows the leader. As a result, each viral mRNA has its own specific 5′UTR in term of length, RNA structure, uORF and Kozak context; each one of these characteristics could affect mRNA expression. In this study, we have measured and compared translational efficiency of each of the ten viral transcripts. Our data show that most of them are very efficiently translated in all translational systems tested. Surprisingly, the gRNA 5′UTR, which is the longest and the most structured, was also the most efficient to initiate translation. This property is conserved in the 5′UTR of SARS-CoV-1 but not in MERS-CoV strain, mainly due to the regulation imposed by the uORF. Interestingly, the translation initiation mechanism on the SARS-CoV-2 gRNA 5′UTR requires the cap structure and the components of the eIF4F complex but showed no dependence in the presence of the poly(A) tail in vitro. Our data strongly suggest that translation initiation on SARS-CoV-2 mRNAs occurs via an unusual cap-dependent mechanism. [ABSTRACT FROM AUTHOR]

Ovarian dysfunction stands as a prevalent contributor to female infertility, with its etiology intertwined with genetic, autoimmune, and environmental factors. Within the ovarian follicles, granulosa cells (GCs) represent the predominant cell population. Alterations in GCs, notably oxidative stress (OS) and the consequential surge in reactive oxygen species (ROS), play pivotal roles in the orchestration of ovarian function. Nrf2aa, a newly identified upstream open reading frame (uORF), is situated within the 5′ untranslated region (5'UTR) of sheep Nrf2 mRNA and is regulated by melatonin, a crucial intrafollicular antioxidant. In this study, we have noted that Nrf2aa has the capacity to encode a peptide and exerts a negative regulatory effect on the translation efficiency (TE) of the Nrf2 CDs region. Further in vitro experiments, we observed that interfering with Nrf2aa can enhance the cellular functionality of GCs under 3-np-induced oxidative stress, while overexpressing Nrf2aa has the opposite effect. Furthermore, overexpression of Nrf2aa counteracts the rescuing effect of melatonin on the cellular functions of GCs under oxidative stress conditions, including estrogen secretion, proliferation, apoptosis, and many more. Finally, we confirmed that Nrf2aa, by regulating the expression of key proteins in the Nrf2/KEAP1 signaling pathway, further modulates the antioxidant levels in GCs. • Identification of a novel sORF, Nrf2aa, located in the 5'UTR of Nrf2 mRNA, whose expression is modulated by melatonin. • Nrf2aa exhibits a reverse regulatory effect on the translation efficiency of Nrf2 in the coding sequence region. • Interfering with Nrf2aa enhances the antioxidant capabilities of sheep granulosa cells under oxidative stress conditions. • Nrf2aa modulates the antioxidant capacity of granulosa cells by regulating the Nrf2/KEAP1 signaling pathway. [ABSTRACT FROM AUTHOR]

The 5' untranslated region (UTR) sequence of eukaryotic mRNAs may contain upstream open reading frames (uORFs), which can regulate translation of the main ORF (mORF). The current model of translational regulation by uORFs posits that when a ribosome scans a mRNA and encounters an uORF, translation of that uORF can prevent ribosomes from reaching the mORF and cause decreased mORF translation. In this study, we first observed that rare variants in the 5' UTR dysregulate maize (Zea mays L.) protein abundance. Upon further investigation, we found that rare variants near the start codon of uORFs can repress or derepressmORF translation, causing allelic changes in protein abundance. This finding holds for common variants as well, and common variants that modify uORF start codons also contribute disproportionately tometabolic and whole-plant phenotypes, suggesting that translational regulation by uORFs serves an adaptive function. These results provide evidence for the mechanisms by which natural sequence variation modulates gene expression, and ultimately, phenotype. [ABSTRACT FROM AUTHOR]

The regulation of protein synthesis plays an important role in the growth and development of all organisms. Upstream open reading frames (uORFs) are commonly found in eukaryotic messenger RNA transcripts and typically attenuate the translation of associated downstream main ORFs (mORFs). Conserved peptide uORFs (CPuORFs) are a rare subset of uORFs, some of which have been shown to conditionally regulate translation by ribosome stalling. Here, we show that Arabidopsis CPuORF19, CPuORF46 and CPuORF47, which are ancient in origin, regulate translation of any downstream ORF, in response to the agriculturally significant environmental signals, heat stress and water limitation. Consequently, these CPuORFs represent a versatile toolkit for inducible gene expression with broad applications. Finally, we note that different classes of CPuORFs may operate during distinct phases of translation, which has implications for the bioengineering of these regulatory factors. Summary statement: We describe three ancient CPuORFs that conditionally regulate translation of any downstream ORF and show the utility of these CPuORFs for rapid responses to agronomically significant stress conditions. [ABSTRACT FROM AUTHOR]

Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNAs (mRNAs) from mouse somites revealed that the 50 leaders of Hox mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer genespecific effects through altered start codon selection stringency [ABSTRACT FROM AUTHOR]

Background: Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood. Methods: We examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs. Results: We provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses. Conclusion: Our data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides. [ABSTRACT FROM AUTHOR]

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila, the sole activating E2F, E2F1, is both required for and sufficient to promote G1fiS progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 50 untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 50UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 50UTR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression. [ABSTRACT FROM AUTHOR]

Polyamines are small organic and basic polycations that perform essential regulatory functions in all living organisms. Fluctuations in polyamine content have been observed to occur during growth, development and under stress conditions, implying that polyamines play pivotal roles in diverse cellular and physiological processes. To achieve polyamine homeostasis, the entire metabolic pathway is subjected to a fine-tuned regulation of its biosynthetic and catabolic genes and enzymes. In this review, we describe and discuss the most important mechanisms implicated in the translational and post-translational regulation of polyamine metabolic enzymes in plants. At the translational level, we emphasize the role of polyamines in the modulation of upstream open reading frame (uORF) activities that control the translation of polyamine biosynthetic and catabolic mRNAs. At the post-translational level, different aspects of the regulation of polyamine metabolic proteins are depicted, such as the proteolytic activation of enzyme precursors, the importance of dimerization in protein stability as well as in protein intracellular localization. • Elements in the 5′-UTR negatively regulate translation of polyamine metabolic mRNAs. • Polyamines modulate uORF activity to control translation of their metabolic mRNAs. • Dimerization impacts the localization and activity of polyamine metabolic proteins. • Polyamine metabolic proteins with PEST sequences are targeted to the 26S-proteasome. • Proteolytic cleavage of enzyme precursors is necessary for polyamine biosynthesis. [ABSTRACT FROM AUTHOR]

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy. [ABSTRACT FROM AUTHOR]

This review describes the current models for how the subunit abundance of the Ndc80 complex, a key kinetochore component, is regulated in budding yeast and metazoan meiosis. The past decades of kinetochore research have established the Ndc80 complex to be a key microtubule interactor and a central hub for regulating chromosome segregation. Recent studies further demonstrate that Ndc80 is the limiting kinetochore subunit that dictates the timing of kinetochore activation in budding yeast meiosis. Here, we discuss the molecular circuits that regulate Ndc80 protein synthesis and degradation in budding yeast meiosis and compare the findings with those from metazoans. We envision the regulatory principles discovered in budding yeast to be conserved in metazoans, thereby providing guidance into future investigations on kinetochore regulation in human health and disease. [ABSTRACT FROM AUTHOR]

Abstract: Translational regulation permeates neuronal function. Nociceptors are sensory neurons responsible for the detection of harmful stimuli. Changes in their activity, termed plasticity, are intimately linked to the persistence of pain. Although inhibitors of protein synthesis robustly attenuate pain-associated behavior, the underlying targets that support plasticity are largely unknown. Here, we examine the contribution of protein synthesis in regions of RNA annotated as noncoding. Based on analyses of previously reported ribosome profiling data, we provide evidence for widespread translation in noncoding transcripts and regulatory regions of mRNAs. We identify an increase in ribosome occupancy in the 5' untranslated regions of the calcitonin gene-related peptide (CGRP/Calca). We validate the existence of an upstream open reading frame (uORF) using a series of reporter assays. Fusion of the uORF to a luciferase reporter revealed active translation in dorsal root ganglion neurons after nucleofection. Injection of the peptide corresponding to the calcitonin gene-related peptide-encoded uORF resulted in pain-associated behavioral responses in vivo and nociceptor sensitization in vitro. An inhibitor of heterotrimeric G protein signaling blocks both effects. Collectively, the data suggest pervasive translation in regions of the transcriptome annotated as noncoding in dorsal root ganglion neurons and identify a specific uORF-encoded peptide that promotes pain sensitization through GPCR signaling. [ABSTRACT FROM AUTHOR]

During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question. [ABSTRACT FROM AUTHOR]

Upstream open reading frames (uORFs) are known to negatively affect translation of the downstream ORF. The regulatory proteins involved in relieving this inhibition are however poorly characterized. In response to cellular stress, eIF2α phosphorylation leads to an inhibition of global protein synthesis, while translation of specific factors such as CHOP is induced. We analyzed a 105‐nt inhibitory uORF in the transcript of human CHOP (huORFchop) and found that overexpression of the zebrafish or human ENDOU poly(U)‐endoribonuclease (Endouc or ENDOU‐1, respectively) increases CHOP mRNA translation also in the absence of stress. We also found that Endouc/ENDOU‐1 binds and cleaves the huORFchop transcript at position 80G‐81U, which induces CHOP translation independently of phosphorylated eIF2α. However, both ENDOU and phospho‐eIF2α are nonetheless required for maximal translation of CHOP mRNA. Increased levels of ENDOU shift a huORFchop reporter as well as endogenous CHOP transcripts from the monosome to polysome fraction, indicating an increase in translation. Furthermore, we found that the uncapped truncated huORFchop‐69‐105‐nt transcript contains an internal ribosome entry site (IRES), facilitating translation of the cleaved transcript. Therefore, we propose a model where ENDOU‐mediated transcript cleavage positively regulates CHOP translation resulting in increased CHOP protein levels upon stress. Specifically, CHOP transcript cleavage changes the configuration of huORFchop thereby releasing its inhibition and allowing the stalled ribosomes to resume translation of the downstream ORF. SYNOPSIS: Heat or ER stress trigger a stress response involving eIF2α phosphorylation and induction of the transcription factor CHOP. Here, stress‐induced poly(U)‐specific endoribonuclease is found to specifically cleave CHOP mRNA, thereby releasing a uORF‐associated inhibitory structure and allowing its selective translation. Zebrafish Endouc and its human orthologue ENDOU positively regulate CHOP translation independently of phosphorylated eIF2α.Both ENDOU and phospho‐eIF2α are required for maximal translation of CHOP upon heat shock or ER stress.ENDOU binds the 105‐nt upstream uORF of the human CHOP transcript and cleaves it at position 80G‐81U.The truncated huORFchop‐81‐105 transcript does not contain IRES activity, while uncapped truncated huORFchop‐69‐105 transcript does. [ABSTRACT FROM AUTHOR]

Micropeptides are small polypeptides coded by small open-reading frames. Progress in computational biology and the analyses of large-scale transcriptomes and proteomes have revealed that mammalian genomes produce a large number of transcripts encoding micropeptides. Many of these have been previously annotated as long noncoding RNAs. The role of micropeptides in cellular homeostasis maintenance has been demonstrated. This review discusses different types of micropeptides as well as methods to identify them, such as computational approaches, ribosome profiling, and mass spectrometry. [ABSTRACT FROM AUTHOR]

Cells utilize transcriptional and posttranscriptional mechanisms to alter gene expression in response to environmental cues. Gene-specific controls, including changing the translation of specific messenger RNAs (mRNAs), provide a rapid means to respond precisely to different conditions. Upstream open reading frames (uORFs) are known to control the translation of mRNAs. Recent studies in bacteria and eukaryotes have revealed the functions of evolutionarily conserved uORF-encoded peptides. Some of these uORF-encoded nascent peptides enable responses to specific metabolites to modulate the translation of their mRNAs by stalling ribosomes and through ribosome stalling may also modulate the level of their mRNAs. In this review, we highlight several examples of conserved uORF nascent peptides that stall ribosomes to regulate gene expression in response to specific metabolites in bacteria, fungi, mammals, and plants. [ABSTRACT FROM AUTHOR]

eIF2α phosphorylation-mediated translational regulation is crucial for global repression of translation by various stresses, including the unfolded protein response (UPR) in eukaryotes. Although translational control during UPR has not been extensively investigated in S. cerevisiae , Hac1-mediated production of long transcripts containing uORFs was shown to repress the translation of histidine triad nucleotide-binding 1 (HNT1) mRNA. The present study showed that uORF3 is required for HNT1 expression, as well as down-regulating HNT1 translation. Translation initiation by uORF3 is inefficient, with uORF3 having a strong Kozak sequence efficiently repressing the translation of HNT1. We propose that leaky scanning of uORF3 is responsible for the downregulation of HNT1 during UPR. • uORF3 is required and sufficient for the down-regulation of HNT1. • uORF3 with strong Kozak sequence efficiently represses translation of HNT1. • Leaky scanning of uORF3 is required for HNT1 down-regulation. [ABSTRACT FROM AUTHOR]

Light is one of the most essential environmental clues for plant growth and morphogenesis. Exposure to blue monochromatic light from darkness is a turning point for plant biological activity, and as a result dramatic changes in gene expression occur. To understand the translational impacts of blue light, we have performed ribosome profiling analysis and called translated open reading frames (ORFs) de novo within not only mRNAs but also non-coding RNAs (ncRNAs). Translation efficiency of 3,823 protein-coding ORFs, such as nuclear chloroplast-related genes, was up-regulated by blue light exposure. Moreover, the translational activation of the microRNA biogenesis-related genes, DCL1 and HYL1 , was induced by blue light. Considering the 3-nucleotide codon periodicity of ribosome footprints, a few hundred short ORFs lying on ncRNAs and upstream ORFs (uORFs) on mRNAs were found that had differential translation status between blue light and dark. uORFs are known to have a negative effect on the expression of the main ORFs (mORFs) on the same mRNAs. Our analysis suggests that the translation of uORFs is likely to be more stimulated than that of the corresponding mORFs, and uORF-mediated translational repression of the mORFs in five genes was alleviated by blue light exposure. With data-based annotation of the ORFs, our analysis provides insights into the translatome in response to environmental changes, such as those involving light. [ABSTRACT FROM AUTHOR]

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC -family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets. [Display omitted] • Ribo-seq reveals widespread translation of non-canonical ORFs in medulloblastoma • High-resolution CRISPR tiling reveals uORF functions in medulloblastoma • The ASNSD1-uORF microprotein controls downstream pathways with the prefoldin-like complex • The ASNSD1-uORF microprotein is necessary for medulloblastoma cell survival Hofman et al. reveal a novel survival mechanism in high-risk childhood medulloblastoma through the translation of non-canonical open reading frames (ORFs). The study uncovers the critical role of these often-overlooked ORFs, particularly the ASNSD1-uORF, in medulloblastoma cell survival, offering new avenues for targeted therapies in pediatric cancer. [ABSTRACT FROM AUTHOR]

• Ribosome profiling was used to study response to environmental stress in fish. • Hypoxia led to profound effect on transcription, translation and protein level in brain. • HIF signaling, autophagy and glycolysis/gluconeogenesis were activated by hypoxia. • Hypoxia disturbed brain function via dysregulated autophagy in yellow catfish. Brain plays a central role in adapting to environmental changes and is highly sensitive to the oxygen level. Although previous studies investigated the molecular response of brain exposure to acute hypoxia in fish, the lack of studies at the translational level hinders further understanding of the regulatory mechanism response to hypoxia from multi-omics levels. Yellow catfish (Pelteobagrus fulvidraco) is an important freshwater aquaculture species; however, hypoxia severely restricts the sustainable development of its breeding industry. In the present study, the transcriptome, translatome, and proteome were integrated to study the global landscapes of yellow catfish brain response to hypoxia. The evidently increased amount of cerebral cortical cells with oedema and pyknotic nuclei has been observed in hypoxia group of yellow catfish. A total of 2750 genes were significantly changed at the translational level. Comparative transcriptional and translational analysis suggested the HIF-1 signaling pathway, autophagy and glycolysis/gluconeogenesis were up-regulated after hypoxia exposure. KEGG enrichment of translational efficiency (TE) differential genes suggested that the lysosome and autophagy were highly enriched. Our result showed that yellow catfish tends to inhibit the TE of genes by increasing the translation of uORFs to adapt to hypoxia. Correlation analysis showed that transcriptome and translatome exhibit higher correlation. In summary, this study demonstrated that hypoxia dysregulated the cerebral function of yellow catfish at the transcriptome, translatome, and proteome, which provides a better understanding of hypoxia adaptation in teleost. [ABSTRACT FROM AUTHOR]

The human inducible nitric oxide synthase (iNOS) gene contains an upstream open reading frame (uORF) in its 5′-untranslated region (5′-UTR) implying a translational regulation of iNOS expression. Transfection experiments in human DLD-1 cells revealed that the uORF although translatable seems not to inhibit the translation start at the bona fide ATG. Our data clearly show that human iNOS translation is cap-dependent and that the 5′-UTR of the iNOS mRNA contains no internal ribosome entry site. Translation of the bona fide coding sequence is most likely mediated by a leaky scanning mechanism. The 5′-UTR is encoded by exon 1 and exon 2 of the iNOS gene with the uORF stop codon located in front of the first intron indicating an involvement of the nonsense mediated RNA decay (NMD) in iNOS regulation. SiRNA-mediated down-regulation of Upf1 resulted in enhanced endogenous cytokine iNOS expression in human DLD-1 cells. Transfection of constructs containing iNOS exon 1, intron 1 and exon 2 in front of a luciferase gene showed a clear effect of the mutation of the uORF-ATG on luciferase reportergene expression. Our data indicate that the uORF in the 5′-UTR sequence of human iNOS gene reduces its expression via the NMD mechanism. • The human iNOS 5′-UTR (encoded by exon 1 and 2) contains an uORF (in front of intron 1). • The uORF is translatable but does not inhibit the translation of the iNOS cds. • Translation of the iNOS cds is likely mediated by a leaky scanning mechanism. • Down-regulation of Upf1 (essential NMD factor) enhanced iNOS expression. • In presence of intron 1 the uORF modulates luciferase reportergene expression. [ABSTRACT FROM AUTHOR]

Upstream open reading frames (uORFs) in 5′ UTRs of eukaryotic mRNAs are increasingly recognized as important elements that regulate cellular protein synthesis. Since uORFs can start from non-AUG codons, an enormous number of potential uORF initiation sites exists in 5′UTRs. However, only a subset of these sites is used and it has been unclear how actual start sites are selected. Studies of the DEAD-box helicase Ded1p from S. cerevisiae show that translation of uORFs with non-AUG initiation codons occurs upstream of mRNA structures that emerge with defective Ded1p. The data designate mRNA structure as important determinant for non-AUG initiation sites of uORFs. Ded1p can control this RNA structure and thereby regulate uORF translation. [ABSTRACT FROM AUTHOR]

Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5'UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in themRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics. [ABSTRACT FROM AUTHOR]

Plants adapt to alterations in light conditions by controlling their gene expression profiles. Expression of light-inducible genes is transcriptionally induced by transcription factors such as HY5. However, few detailed analyses have been carried out on the control of transcription start sites (TSSs). Of the various wavelengths of light, it is blue light (BL) that regulates physiological responses such as hypocotyl elongation and flowering time. To understand how gene expression is controlled not only by transcript abundance but also by TSS selection, we examined genome-wide TSS profiles in Arabidopsis seedlings after exposure to BL irradiation following initial growth in the dark. Thousands of genes use multiple TSSs, and some transcripts have upstream ORFs (uORFs) that take precedence over the main ORF (mORF) encoding proteins. The uORFs often function as translation inhibitors of the mORF or as triggers of nonsense-mediated mRNA decay (NMD). Transcription from TSSs located downstream of the uORFs in 220 genes is enhanced by BL exposure. This type of regulation is found in HY5 and HYH, major regulators of light-dependent gene expression. Translation efficiencies of the genes showing enhanced usage of these TSSs increased upon BL exposure. We also show that transcripts from TSSs upstream of uORFs in 45 of the 220 genes, including HY5, accumulated in a mutant of NMD. These results suggest that BL controls gene expression not only by enhancing transcriptions but also by choosing the TSS, and transcripts from downstream TSSs evade uORF-mediated inhibition to ensure high expression of light-regulated genes. [ABSTRACT FROM AUTHOR]

Eukaryotic cells evolved highly complex and accurate protein synthesis machinery that is finely tuned by various signaling pathways. Dysregulation of translation is a hallmark of many diseases, including cancer, and thus pharmacological approaches to modulate translation become very promising. While there has been much progress in our understanding of mammalian mRNA-specific translation control, surprisingly, relatively little is known about whether and how the protein components of the translation machinery shape translation of their own mRNAs. Here we analyze mammalian mRNAs encoding components of the translation initiation machinery for potential regulatory features such as 5′TOP motifs, TISU motifs, poor start codon nucleotide context and upstream open reading frames. [ABSTRACT FROM AUTHOR]

Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control. [ABSTRACT FROM AUTHOR]

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions ( UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'- UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria. [ABSTRACT FROM AUTHOR]

Background: With the development of rapid and inexpensive DNA sequencing, the genome sequences of more than 100 fungal species have been made available. This dataset provides an excellent resource for comparative genomics analyses, which can be used to discover genetic elements, including noncoding RNAs (ncRNAs). Bioinformatics tools similar to those used to uncover novel ncRNAs in bacteria, likewise, should be useful for searching fungal genomic sequences, and the relative ease of genetic experiments with some model fungal species could facilitate experimental validation studies. Results: We have adapted a bioinformatics pipeline for discovering bacterial ncRNAs to systematically analyze many fungal genomes. This comparative genomics pipeline integrates information on conserved RNA sequence and structural features with alternative splicing information to reveal fungal RNA motifs that are candidate regulatory domains, or that might have other possible functions. A total of 15 prominent classes of structured ncRNA candidates were identified, including variant HDV self-cleaving ribozyme representatives, atypical snoRNA candidates, and possible structured antisense RNA motifs. Candidate regulatory motifs were also found associated with genes for ribosomal proteins, S-adenosylmethionine decarboxylase (SDC), amidase, and HexA protein involved in Woronin body formation. We experimentally confirm that the variant HDV ribozymes undergo rapid self-cleavage, and we demonstrate that the SDC RNA motif reduces the expression of SAM decarboxylase by translational repression. Furthermore, we provide evidence that several other motifs discovered in this study are likely to be functional ncRNA elements. Conclusions: Systematic screening of fungal genomes using a computational discovery pipeline has revealed the existence of a variety of novel structured ncRNAs. Genome contexts and similarities to known ncRNA motifs provide strong evidence for the biological and biochemical functions of some newly found ncRNA motifs. Although initial examinations of several motifs provide evidence for their likely functions, other motifs will require more in-depth analysis to reveal their functions. [ABSTRACT FROM AUTHOR]

A large body of evidence indicates that genome annotation pipelines have biased our view of coding sequences because they generally undersample small proteins and peptides. The recent development of genome-wide translation profiling reveals the prevalence of small/short open reading frames (smORFs or sORFs), which are scattered over all classes of transcripts, including both mRNAs and presumptive long noncoding RNAs. Proteomic approaches further confirm an unexpected variety of smORF-encoded peptides (SEPs), representing an overlooked reservoir of bioactive molecules. Indeed, functional studies in a broad range of species from yeast to humans demonstrate that SEPs can harbor key activities for the control of development, differentiation, and physiology. Here we summarize recent advances in the discovery and functional characterization of smORF/SEPs and discuss why these small players can no longer be ignored with regard to genome function. [ABSTRACT FROM AUTHOR]

Background Campomelic dysplasia ( CD) is a semilethal developmental disorder caused by mutations in and around SOX9. CD is characterized by multiple skeletal malformations including bending (campomelia) of long bones. Surviving patients frequently have the acampomelic form of CD ( ACD). Methods This is a single case report on a patient with clinical and radiological features of ACD who has no mutation in the SOX9 protein-coding sequence nor a translocation with breakpoint in the SOX9 regulatory domain. We include functional studies of the novel mutant protein in vitro and in cultured cells. Results The patient was found to have a de novo heterozygous mutation c.-185G>A in the SOX9 5′ UTR. The mutation creates an upstream translation start codon, uAUG, with a much better fit of its flanking sequence to the Kozak consensus than the wild-type AUG. By in vitro transcription-translation and transient transfection into COS-7 cells, we show that the uAUG leads to translation of a short peptide from a reading frame that terminates just after the wild-type AUG start codon. This results in reduced translation of the wild-type protein, compatible with the milder phenotype of the patient. Conclusion Findings support the notion that more mildly affected, surviving CD/ ACD patients carry mutant SOX9 alleles with residual expression of SOX9 wild-type protein. Although rarely described in human genetic disease and for the first time here for CD, mutations creating upstream AUG codons may be more common than generally assumed. [ABSTRACT FROM AUTHOR]

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5'-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. Alternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression. • Alternative SERPINA1 long mRNA translation initiates at AUG-2. • SERPINA1 uORFs regulate translation from AUG-1, not AUG-2. • Non-secretory AAT isoform-eGFP fusion product exhibits nuclear-cytoplasmic shuttling. • Putative endogenous AAT isoform enriches the nuclear speckles. • Alternative SERPINA1 translation may expand the functional serpin's repertoire. [ABSTRACT FROM AUTHOR]

• BCL2L2 and nine alternative splicing transcripts were cloned and identified in pigs. • One SP1 and two C/EBPα binding sites were characterized in the promoter. • Alternative splicing in 5′ UTR regulates the expression of BCL2L2. • Upstream opening reading frame represses the expression of BCL2L2. B cell lymphoma-2-like 2 (BCL2L2), an important regulator of apoptosis, plays vital roles in several physiological processes, as revealed by studies in humans and mice. However, reports on pig BCL2L2 are few, and the encoding gene has not been identified experimentally. This study was designed to clone the porcine BCL2L2 gene and its alternative splicing (AS) transcripts using molecular biological techniques and to analyze the regulatory mechanisms underlying transcription and translation. The BCL2L2 cDNA (V1) was 807 bp in length and encoded a polypeptide of 193 aa containing four BCL-2 homology domains. A total of nine AS transcripts were obtained, among which V2 and V3 differed from V1 in the 5′ untranslated region (UTR). The core promoter was mapped to a range of −1102 to −759 bp (the first nucleotide of the start codon was designated as +1). There were several functional cis -elements, including one SP1 and two C/EBPα binding sites at around −759 bp. AS in the 5′ UTR is involved in the regulation of gene expression, as revealed by dual-luciferase reporter and western blot analysis, and the secondary structure of the 5′ UTRs may be the reason for the differential expression of V1–3. At the same time, an upstream open reading frame (ORF) existed in each of the three 5′ UTRs, was found to repress the expression of the main ORF. Additionally, the roles of porcine BCL2L2 in cell proliferation and apoptosis were preliminarily analyzed. The results will contribute to further characterizing the role of BCL2L2. [ABSTRACT FROM AUTHOR]