Downstream Open Reading Frame Research Articles

Efficiency of potato genome editing: Targeted mutation on the genes involved in starch biosynthesis using the CRISPR/dMac3-Cas9 system.

Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

Analysis of COL1A1 and COL1A2 gene variants in two fetuses with osteogenesis imperfecta

To explore the genetic basis of two fetuses with an osteogenesis imperfecta (OI) phenotype. Two fetuses diagnosed at the Affiliated Hospital of Weifang Medical College respectively on June 11, 2021 and October 16, 2021 were selected as the study subjects. Clinical data of the fetuses were collected. Amniotic fluid samples of the fetuses and peripheral blood samples of their pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing (WES) and Sanger sequencing were carried out to identify the candidate variants. Minigene splicing reporter analysis was used to validate the variant which may affect the pre-mRNA splicing. For fetus 1, ultrasonography at 17+6 weeks of gestation had revealed shortening of bilateral humerus and femurs by more than two weeks, in addition with multiple fractures and angular deformities of long bones. WES revealed that fetus 1 had harbored a heterozygous c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in exon 49 of the COL1A1 gene (NM_000088.4). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), it was classified as a pathogenic variant (PVS1+PS2+PM2_Supporting) for disrupting the downstream open reading frame resulting in premature translational termination, being de novo in origin, and lacking records in the population and disease databases.For fetus 2, ultrasonography at 23 weeks of gestation also revealed shortening of bilateral humerus and femurs by one and four weeks, respectively, in addition with bending of bilateral femurs, tibias and fibulas. Fetus 2 had harbored a heterozygous c.1557+3A>G variant in intron 26 of the COL1A2 gene (NM_000089.4). Minigene experiment showed that it has induced skipping of exon 26 from the COL1A2 mRNA transcript, resulting in an in-frame deletion (c.1504_1557del) of the COL1A2 mRNA transcript. The variant was inherited from its father and had been previously reported in a family with OI type 4. It was therefore classified as a pathogenic variant (PS3+PM1+PM2_Supporting+PP3+PP5). The c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in the COL1A1 gene and c.1557+3A>G variant in the COL1A2 gene probably underlay the disease in the two fetuses. Above findings not only have enriched the mutational spectrum of OI, but also shed light on the correlation between its genotype and phenotype and provided a basis for genetic counseling and prenatal diagnosis for the affected pedigrees.

Dynamic Translational Landscape Revealed by Genome-Wide Ribosome Profiling under Drought and Heat Stress in Potato.

The yield and quality of potatoes, an important staple crop, are seriously threatened by high temperature and drought stress. In order to deal with this adverse environment, plants have evolved a series of response mechanisms. However, the molecular mechanism of potato's response to environmental changes at the translational level is still unclear. In this study, we performed transcriptome- and ribosome-profiling assays with potato seedlings growing under normal, drought, and high-temperature conditions to reveal the dynamic translational landscapes for the first time. The translational efficiency was significantly affected by drought and heat stress in potato. A relatively high correlation (0.88 and 0.82 for drought and heat stress, respectively) of the fold changes of gene expression was observed between the transcriptional level and translational level globally based on the ribosome-profiling and RNA-seq data. However, only 41.58% and 27.69% of the different expressed genes were shared by transcription and translation in drought and heat stress, respectively, suggesting that the transcription or translation process can be changed independently. In total, the translational efficiency of 151 (83 and 68 for drought and heat, respectively) genes was significantly changed. In addition, sequence features, including GC content, sequence length, and normalized minimal free energy, significantly affected the translational efficiencies of genes. In addition, 28,490 upstream open reading frames (uORFs) were detected on 6463 genes, with an average of 4.4 uORFs per gene and a median length of 100 bp. These uORFs significantly affected the translational efficiency of downstream major open reading frames (mORFs). These results provide new information and directions for analyzing the molecular regulatory network of potato seedlings in response to drought and heat stress.

The translational landscape of bread wheat during grain development.

The dynamics of gene expression in crop grains has typically been investigated at the transcriptional level. However, this approach neglects translational regulation, a widespread mechanism that rapidly modulates gene expression to increase the plasticity of organisms. Here, we performed ribosome profiling and polysome profiling to obtain a comprehensive translatome data set of developing bread wheat (Triticum aestivum) grains. We further investigated the genome-wide translational dynamics during grain development, revealing that the translation of many functional genes is modulated in a stage-specific manner. The unbalanced translation between subgenomes is pervasive, which increases the expression flexibility of allohexaploid wheat. In addition, we uncovered widespread previously unannotated translation events, including upstream open reading frames (uORFs), downstream open reading frames (dORFs), and open reading frames (ORFs) in long noncoding RNAs, and characterized the temporal expression dynamics of small ORFs. We demonstrated that uORFs act as cis-regulatory elements that can repress or even enhance the translation of mRNAs. Gene translation may be combinatorially modulated by uORFs, dORFs, and microRNAs. In summary, our study presents a translatomic resource that provides a comprehensive and detailed overview of the translational regulation in developing bread wheat grains. This resource will facilitate future crop improvements for optimal yield and quality.

Translational buffering by ribosome stalling in upstream open reading frames.

Upstream open reading frames (uORFs) are present in over half of all human mRNAs. uORFs can potently regulate the translation of downstream open reading frames through several mechanisms: siphoning away scanning ribosomes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. Here, we performed systematic measurements on the uORF-containing 5' UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF protein expression when ribosome loading onto the mRNA is reduced. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. We identify several human uORFs that repress main ORF protein expression via a similar terminal diproline motif. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions.

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

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

The dark proteome: translation from noncanonical open reading frames.

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

Rebirth of the translational machinery: The importance of recycling ribosomes.

Translation of the genetic code occurs in a cycle where ribosomes engage mRNAs, synthesize protein, and then disengage in order to repeat the process again. The final part of this process-ribosome recycling, where ribosomes dissociate from mRNAs-involves a complex molecular choreography of specific protein factors to remove the large and small subunits of the ribosome in a coordinated fashion. Errors in this process can lead to the accumulation of ribosomes at stop codons or translation of downstream open reading frames (ORFs). Ribosome recycling is also critical when a ribosome stalls during the elongation phase of translation and must be rescued to allow continued translation of the mRNA. Here we discuss the molecular interactions that drive ribosome recycling, and their regulation in the cell. We also examine the consequences of inefficient recycling with regards to disease, and its functional roles in synthesis of novel peptides, regulation of gene expression, and control of mRNA-associated proteins. Alterations in ribosome recycling efficiency have the potential to impact many cellular functions but additional work is needed to understand how this regulatory power is utilized.

Disorder at the Start: The Contribution of Dysregulated Translation Initiation to Cancer Therapy Resistance.

Translation of cellular RNA to protein is an energy-intensive process through which synthesized proteins dictate cellular processes and function. Translation is regulated in response to extracellular effectors and availability of amino acids intracellularly. Most eukaryotic mRNA rely on the methyl 7-guanosine (m7G) nucleotide cap to recruit the translation machinery, and the uncoupling of translational control that occurs in tumorigenesis plays a significant role in cancer treatment response. This article provides an overview of the mammalian translation initiation process and the primary mechanisms by which it is regulated. An outline of how deregulation of initiation supports tumorigenesis and how initiation at a downstream open reading frame (ORF) of Tousled-like kinase 1 (TLK1) leads to treatment resistance is discussed.

A deterministic model for non-monotone relationship between translation of upstream and downstream open reading frames.

Totally asymmetric simple exclusion process (TASEP) modelling was shown to offer a parsimonious explanation for the experimentally confirmed ability of a single upstream open reading frames (uORFs) to upregulate downstream translation during the integrated stress response. As revealed by numerical simulations, the model predicts that reducing the density of scanning ribosomes upstream of certain uORFs increases the flow of ribosomes downstream. To gain a better insight into the mechanism which ensures the non-monotone relation between the upstream and downstream flows, in this work, we propose a phenomenological deterministic model approximating the TASEP model of the translation process. We establish the existence of a stationary solution featuring the decreasing density along the uORF for the deterministic model. Further, we find an explicit non-monotone relation between the upstream ribosome density and the downstream flow for the stationary solution in the limit of increasing uORF length and increasingly leaky initiation. The stationary distribution of the TASEP model, the stationary solution of the deterministic model and the explicit limit are compared numerically.

Tissue- and stage-specific landscape of the mouse translatome.

The current understanding of how overall principles of translational control govern the embryo-to-adult transition in mammals is still far from comprehensive. Herein we profiled the translatomes and transcriptomes of six tissues from the mice at embryonic and adult stages and presented the first report of tissue- and stage-specific translational landscape in mice. We quantified the extent of gene expression divergence among different expression layers, tissues and stages, detected significant changes in gene composition and function underlying these divergences and revealed the changing architecture of translational regulation. We further showed that dynamic translational regulation can be largely achieved via modulation of translational efficiency. Translational efficiency could be altered by alternative splicing (AS), upstream and downstream open reading frames (uORFs and dORFs). We revealed AS-mediated translational repression that was exerted in an event type-dependent manner. uORFs and dORFs exhibited mutually exclusive usage and the opposing effects of translational regulation. Furthermore, we discovered many novel microproteins encoded by long noncoding RNAs and demonstrated their regulatory potential and functional relevance. Our data and analyses will facilitate a better understanding of the complexity of translation and translational regulation across tissue and stage spectra and provide an important resource to the translatome research community.

Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium meliloti selB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

EIF4G-driven translation initiation of downstream ORFs in mammalian cells.

Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3′ untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3′ UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3′ UTRs. The 3′ UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3′ UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3′ UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3′ UTRs of mRNAs may encode various products.

Translation of small downstream ORFs enhances translation of canonical main open reading frames.

In addition to canonical open reading frames (ORFs), thousands of translated small ORFs (containing less than 100 codons) have been identified in untranslated mRNA regions (UTRs) across eukaryotes. Small ORFs in 5′ UTRs (upstream (u)ORFs) often repress translation of the canonical ORF within the same mRNA. However, the function of translated small ORFs in the 3′ UTRs (downstream (d)ORFs) is unknown. Contrary to uORFs, we find that translation of dORFs enhances translation of their corresponding canonical ORFs. This translation stimulatory effect of dORFs depends on the number of dORFs, but not the length or peptide they encode. We propose that dORFs represent a new, strong, and universal translation regulatory mechanism in vertebrates.

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

Messenger RNAs with large numbers of upstream open reading frames are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum.

The genome of Plasmodium falciparum has one of the most skewed base-pair compositions of any eukaryote, with an AT content of 80-90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 11 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream open reading frame (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation.In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case, the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to the codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole-genome data was analysed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

Regulation of mRNA translation by a photoriboswitch.

Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)-a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes.

UORFlight: a vehicle toward uORF-mediated translational regulation mechanisms in eukaryotes.

Upstream open reading frames (uORFs) are prevalent in eukaryotic mRNAs. They act as a translational control element for precisely tuning the expression of the downstream major open reading frame (mORF). uORF variation has been clearly associated with several human diseases. In contrast, natural uORF variants in plants have not ever been identified or linked with any phenotypic changes. The paucity of such evidence encouraged us to generate this database-uORFlight (http://uorflight.whu.edu.cn). It facilitates the exploration of uORF variation among different splicing models of Arabidopsis and rice genes. Most importantly, users can evaluate uORF frequency among different accessions at the population scale and find out the causal single nucleotide polymorphism (SNP) or insertion/deletion (INDEL), which can be associated with phenotypic variation through database mining or simple experiments. Such information will help to make hypothesis of uORF function in plant development or adaption to changing environments on the basis of the cognate mORF function. This database also curates plant uORF relevant literature into distinct groups. To be broadly interesting, our database expands uORF annotation into more species of fungus (Botrytis cinerea and Saccharomyces cerevisiae), plant (Brassica napus, Glycine max, Gossypium raimondii, Medicago truncatula, Solanum lycopersicum, Solanum tuberosum, Triticum aestivum and Zea mays), metazoan (Caenorhabditis elegans and Drosophila melanogaster) and vertebrate (Homo sapiens, Mus musculus and Danio rerio). Therefore, uORFlight will light up the runway toward how uORF genetic variation determines phenotypic diversity and advance our understanding of translational control mechanisms in eukaryotes.

Impacts of uORF codon identity and position on translation regulation.

Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5′ mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.

Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature.

The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.