Translation Of Open Reading Frame Research Articles

MisORFPred: A Novel Method to Mine Translatable sORFs in Plant Pri-miRNAs Using Enhanced Scalable k-mer and Dynamic Ensemble Voting Strategy.

The primary microRNAs (pri-miRNAs) have been observed to contain translatable small open reading frames (sORFs) that can encode peptides as an independent element. Relevant studies have proven that those of sORFs are of significance in regulating the expression of biological traits. The existing methods for predicting the coding potential of sORFs frequently overlook this data or categorize them as negative samples, impeding the identification of additional translatable sORFs in pri-miRNAs. In light of this, a novel method named misORFPred has been proposed. Specifically, an enhanced scalable k-mer (ESKmer) that simultaneously integrates the composition information within a sequence and distance information between sequences is designed to extract the nucleotide sequence features. After feature selection, the optimal features and several machine learning classifiers are combined to construct the ensemble model, where a newly devised dynamic ensemble voting strategy (DEVS) is proposed to dynamically adjust the weights of base classifiers and adaptively select the optimal base classifiers for each unlabeled sample. Cross-validation results suggest that ESKmer and DEVS are essential for this classification task and could boost model performance. Independent testing results indicate that misORFPred outperforms the state-of-the-art methods. Furthermore, we execute misORFPerd on the genomes of various plant species and perform a thorough analysis of the predicted outcomes. Taken together, misORFPred is a powerful tool for identifying the translatable sORFs in plant pri-miRNAs and can provide highly trusted candidates for subsequent biological experiments.

Structural basis for translational control by the human 48S initiation complex.

The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome.

RESC14 and RESC8 cooperate to mediate RESC function and dynamics during trypanosome RNA editing.

Mitochondrial transcripts in Trypanosoma brucei require extensive uridine insertion/deletion RNA editing to generate translatable open reading frames. The RNA editing substrate binding complex (RESC) serves as the scaffold that coordinates the protein-protein and protein-RNA interactions during editing. RESC broadly contains two modules termed the guide RNA binding complex (GRBC) and the RNA editing mediator complex (REMC), as well as organizer proteins. How the protein and RNA components of RESC dynamically interact to facilitate editing is not well understood. Here, we examine the roles of organizer proteins, RESC8 and RESC14, in facilitating RESC dynamics. High-throughput sequencing of editing intermediates reveals an overlapping RESC8 and RESC14 function during editing progression across multiple transcripts. Blue native PAGE analysis demonstrates that RESC14 is essential for incorporation of RESC8 into a large RNA-containing complex, while RESC8 is important in recruiting a smaller ribonucleoprotein complex (RNP) to this large complex. Proximity labeling shows that RESC14 is important for stable RESC protein-protein interactions, as well as RESC-RECC associations. Together, our data support a model in which RESC14 is necessary for assembly of editing competent RESC through recruitment of an RNP containing RESC8, GRBC and gRNA to REMC and mRNA.

Comparison of software packages for detecting unannotated translated small open reading frames by Ribo-seq.

Accurate and comprehensive annotation of microprotein-coding small open reading frames (smORFs) is critical to our understanding of normal physiology and disease. Empirical identification of translated smORFs is carried out primarily using ribosome profiling (Ribo-seq). While effective, published Ribo-seq datasets can vary drastically in quality and different analysis tools are frequently employed. Here, we examine the impact of these factors on identifying translated smORFs. We compared five commonly used software tools that assess open reading frame translation from Ribo-seq (RibORFv0.1, RibORFv1.0, RiboCode, ORFquant, and Ribo-TISH) and found surprisingly low agreement across all tools. Only ~2% of smORFs were called translated by all five tools, and ~15% by three or more tools when assessing the same high-resolution Ribo-seq dataset. For larger annotated genes, the same analysis showed ~74% agreement across all five tools. We also found that some tools are strongly biased against low-resolution Ribo-seq data, while others are more tolerant. Analyzing Ribo-seq coverage revealed that smORFs detected by more than one tool tend to have higher translation levels and higher fractions of in-frame reads, consistent with what was observed for annotated genes. Together these results support employing multiple tools to identify the most confident microprotein-coding smORFs and choosing the tools based on the quality of the dataset and the planned downstream characterization experiments of the predicted smORFs.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

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.

Ribosome Profiling of Plants.

Translation is a key step in control of gene expression, yet most analyses of global responses to a stimulus focus on transcription and the transcriptome. For RNA viruses in particular, which have no DNA-templated transcriptional control, control of viral and host translation is crucial. Here, we describe the method of ribosome profiling (ribo-seq) in plants, applied to virus infection. Ribo-seq is a deep sequencing technique that reveals the translatome by presenting a snapshot of the positions and relative amounts of translating ribosomes on all mRNAs in the cell. In contrast to RNA-seq, a crude cell extract is first digested with ribonuclease to degrade all mRNA not protected by a translating 80S ribosome. The resulting ribosome-protected fragments (RPFs) are deep sequenced. The number of reads mapping to a specific mRNA compared to the standard RNA-seq reads reveals the translational efficiency of that mRNA. Moreover, the precise positions of ribosome pause sites, previously unknown translatable open reading frames, and noncanonical translation events can be characterized quantitatively using ribo-seq. As this technique requires meticulous technique, here we present detailed step-by-step instructions for cell lysate preparation by flash freezing of samples, nuclease digestion of cell lysate, monosome collection by sucrose cushion ultracentrifugation, size-selective RNA extraction and rRNA depletion, library preparation for sequencing and finally quality control of sequenced data. These experimental methods apply to many plant systems, with minor nuclease digestion modifications depending on the plant tissue and species. This protocol should be valuable for studies of plant virus gene expression, and the global translational response to virus infection, or any other biotic or abiotic stress, by the host plant.

In-depth characterization and identification of translatable lncRNAs

Long non-coding RNAs (LncRNAs) are non-protein coding transcripts more than 200 nucleotides in length. Deep sequencing technologies have unveiled lncRNAs can harbor translatable short open reading frames (sORFs). Yet the regulatory mechanisms governing lncRNA translation events remain poorly understood. Here, we exhaustively detected the sequence, functional element, and structure features relevant to lncRNA translation in human. Extensive identification and analysis reveal that translatable lncRNAs contain richer protein-coding related sequence features, cap-dependent and cap-independent translation initiation mechanisms, and more stable secondary structures, as compared to untranslatable lncRNAs. These findings strongly support lncRNAs serve as a repository for the production of new small peptides. Based on the feature fusion affecting translation and the extreme gradient boosting (XGBoost) algorithm, we developed the first computational tool that dedicated for predicting translatable lncRNAs, named TransLncPred. Benchmark experimental results show that our method outperforms several state-of-the-art RNA coding potential prediction tools on the same training and testing datasets. The 100-time 10-fold cross-validation tests also demonstrate that regulatory element-derived features, especially N7-methylguanosine (m7G) and internal ribosome entry site (IRES), contribute to the improvement in predictive performance.

A novel ambigrammatic mycovirus, PsV5, works hand in glove with wheat stripe rust fungus to facilitate infection

Here we describe a novel narnavirus, Puccinia striiformis virus 5 (PsV5), from the devastating wheat stripe rust fungus P. striiformis f. sp. tritici (Pst). The genome of PsV5 contains two predicted open reading frames (ORFs) that largely overlap on reverse strands: an RNA-dependent RNA polymerase (RdRp) and a reverse-frame ORF (rORF) with unknown function. Protein translations of both ORFs were demonstrated by immune technology. Transgenic wheat lines overexpressing PsV5 (RdRp-rORF), RdRp ORF, or rORF were more susceptible to Pst infection, whereas PsV5-RNA interference (RNAi) lines were more resistant. Overexpression of PsV5 (RdRp-rORF), RdRp ORF, or rORF in Fusarium graminearum also boosted fungal virulence. We thus report a novel ambigrammatic mycovirus that promotes the virulence of its fungal host. The results are a significant addition to our understanding of virosphere diversity and offer insights for sustainable wheat rust disease control.

470-P: Mitochondrial Long Noncoding RNA and the Compromised Electron Transport Chain Machinery in Diabetic Retinopathy

Purpose: Diabetic retinopathy has a complex pathogenesis, and mitochondrial damage is considered to play a central role. Diabetes dysfunctions the mitochondria and damages their DNA (mtDNA) , decreasing the transcription of mtDNA-encoded genes including Cytochrome B (CYTB) , which is important for the functioning of complex III of the electron transport chain system (ETC) . This propagate the vicious cycle of free radicals. Diabetes also decreases mtDNA-encoded long non-coding RNA Cytochrome B (LncCytB) . Although long noncoding RNAs have no open reading frame for translation, they can bind to the DNA or RNA in a sequence specific manner and alter the gene expression. Our aim was to investigate the role of LncCytB in regulating CYTB-ETC system in diabetic retinopathy. Methods: Human retinal endothelial cells, incubated in 5mM (normal) or 20mM (high) D-glucose, were used to quantify the expressions of LncCytB (by strand-specific PCR) and CYTB (qRT-PCR) . The binding of LncCytB at CYTB promoter was determined by Chromatin isolation by RNA Purification method. Role of LncCytB in regulating CYTB expression and mitochondrial damage was performed in the cells overexpressing LncCytB. Results: Compared to cells in normal glucose, LncCytB and CYTB levels were decreased by over 50% in the cells exposed to high glucose, the binding of LncCytB at CYTB promoter was reduced by ∼40% and the activity of the complex III was inhibited. Overexpression of LncCytB ameliorated glucose-induced decrease in CYTB gene transcripts, and increase in mtDNA damage and in mitochondrial reactive oxygen species. Conclusions: Due to decreased interactions between LncCytB-CYTB in hyperglycemic milieu, CYTB transcripts are decreased, and this compromises the ETC system, resulting in increased mitochondrial ROS. Thus, regulating LncCytB levels in diabetes could halt the self-propagating cycle of free radicals, and prevent the development of diabetic retinopathy. Funding EY014370, EY017313, and EY022230

Small ORFs as New Regulators of Pri-miRNAs and miRNAs Expression in Human and Drosophila.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.

Combining Protein Expression and Molecular Data Improves Mutation Characterization of Dystrophinopathies.

Duchenne and Becker muscular dystrophy are X-linked recessive inherited disorders characterized by progressive weakness due to skeletal muscle degeneration. Different mutations in the DMD gene, which encodes for dystrophin protein, are responsible for these disorders. The aim of our study was to investigate the relationship between type, size, and location of the mutation that occurs in the DMD gene and their effect on dystrophin protein expression in a cohort of 40 male dystrophinopathy patients and nine females, possible carriers. We evaluated the expression of dystrophin by immunofluorescence and immunoblotting. The mutational spectrum of the DMD gene was established by MLPA for large copy number variants, followed by HRM analysis for point mutations and sequencing of samples with an abnormal melting profile. MLPA revealed 30 deletions (75%) and three duplications (7.5%). HRM analysis accounted for seven-point mutations (17.5%). We also report four novel small mutations (c. 8507G>T, c.3021delG, c.9563_9563+1insAGCATGTTTATGATACAGCA, c.7661-60T>A) in DMD gene. Our work shows that the DNA translational open reading frame and the location of the mutation both influence the expression of dystrophin and disease severity phenotype. The proposed algorithm used in this study demonstrates its accuracy for the characterization of dystrophinopathy patients.

Abstract CC01-02: Novel algorithm discovers up to 35% more epitopes translated from non-coding regions in cold tumours

Abstract Currently, most neoantigen pipelines often focus on the detection of neoantigens derived from mutations in the coding regions of the genome. However, in some cancer indications, the number of mutations detectable in tumours can be very low (low tumour mutational burden). This limits the number of actionable neoantigens and results in so-called “cold” tumours. In these cases, non-canonical neoantigens resulting from alterations in non-coding regions of the human genome could represent a high potential alternative for treatment. Indeed, recent research has revealed that previously presumed non-coding regions of the human genome, such as long non-coding RNAs (lncRNAs), can contain translatable small open reading frames (smORFs) generating micropeptides. Some of these micropeptides have already been shown to be involved in cancer development, but these small peptides could also represent a high potential source of non-canonical neoantigens for personalised therapy. Here, we present smORFin, a machine learning algorithm specifically trained to identify smORFs in transcripts and to assess their coding potential. While most tools are focused on longer sequences, smORFin is specifically developed to target small ORFs (<303 nucleotides). Furthermore, smORFin also accounts for smORFs with alternative initiation codons, thereby improving its sensitivity for the detection of novel unannotated smORFs. As a result, the smORFin model reaches a precision of 0.98 and an accuracy of 0.95 on its testing dataset. Using this new prediction tool, a library of human smORFs was assembled, the so-called smORFeome. This library of smORFs, and their associated proteins, was evaluated as a reference for spectrum to peptide matching in mass spectrometry data (MS) analysis. Indeed, the evaluation of seven MS samples revealed and validated the presence of smORFeome-related micropeptides and HLA-Iassociated epitopes originating from smORFs. The impact of mutations in allegedly non-coding regions of tumour genomes and its influence on the neoantigen repertoire, was evaluated through integration of smORFin in a neoantigen identification pipeline targeting lncRNA-derived mutated epitopes; lncRNeos. It was observed that these epitopes only represent a minor fraction of the total neoantigen load. Strikingly, when only focusing on tumours with a low neoantigen load, lncRNeos represented up to 27% of the total neoantigen load. This indicates that for tumours with a low TMB, and therefore with a low neoantigen load, lncRNeos allows to significantly expand the neoantigen repertoire. Biological in vivo/in vitro validation remains necessary to assess the existence, presentation, and actionability of lncRNeos. Citation Format: Lena Pfitzer, Lore Van Oudenhove, Cedric Bogaert, Bruno Fant. Novel algorithm discovers up to 35% more epitopes translated from non-coding regions in cold tumours [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC01-02.

Nuclear Genome-Encoded Long Noncoding RNAs and Mitochondrial Damage in Diabetic Retinopathy.

Retinal mitochondria are damaged in diabetes-accelerating apoptosis of capillary cells, and ultimately, leading to degenerative capillaries. Diabetes also upregulates many long noncoding RNAs (LncRNAs), including LncMALAT1 and LncNEAT1. These RNAs have more than 200 nucleotides and no open reading frame for translation. LncMALAT1 and LncNEAT1 are encoded by nuclear genome, but nuclear-encoded LncRNAs can also translocate in the mitochondria. Our aim was to investigate the role of LncMALAT1 and LncNEAT1 in mitochondrial homeostasis. Using human retinal endothelial cells, the effect of high glucose on LncMALAT1 and LncNEAT1 mitochondrial localization was examined by RNA fluorescence in situ hybridization. The role of these LncRNAs in mitochondrial membrane potential (by JC-I staining), mtDNA integrity (by extended length PCR) and in protective mtDNA nucleoids (by SYBR green staining) was examined in MALAT1- or NEAT1-siRNA transfected cells. High glucose increased LncMALAT1 and LncNEAT1 mitochondrial expression, and MALAT1-siRNA or NEAT1-siRNA ameliorated glucose-induced damage to mitochondrial membrane potential and mtDNA, and prevented decrease in mtDNA nucleoids. Thus, increased mitochondrial translocation of LncMALAT1 or LncNEAT1 in a hyperglycemic milieu plays a major role in damaging the mitochondrial structural and genomic integrity. Regulation of these LncRNAs can protect mitochondrial homeostasis, and ameliorate formation of degenerative capillaries in diabetic retinopathy.

Cellular Adaptations to Cytoplasmic Mg2+ Limitation.

Mg2+ is the most abundant divalent cation in living cells. It is essential for charge neutralization, macromolecule stabilization, and the assembly and activity of ribosomes and as a cofactor for enzymatic reactions. When experiencing low cytoplasmic Mg2+, bacteria adopt two main strategies: They increase the abundance and activity of Mg2+ importers and decrease the abundance of Mg2+-chelating ATP and rRNA. These changes reduce regulated proteolysis by ATP-dependent proteases and protein synthesis in a systemic fashion. In many bacterial species, the transcriptional regulator PhoP controls expression of proteins mediating these changes. The 5' leader region of some mRNAs responds to low cytoplasmic Mg2+ or to disruptions in translation of open reading frames in the leader regions by furthering expression of the associated coding regions, which specify proteins mediating survival when the cytoplasmic Mg2+ concentration is low. Microbial species often utilize similar adaptation strategies to cope with low cytoplasmic Mg2+ despite relying on different genes to do so.

Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells.

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

Ribosomal profiling as a tool for studying translation in plants: main results, problems and future prospects

Экспрессию эукариотических генов можно регулировать на нескольких этапах, включая трансляцию мРНК. Известно, что структура мРНК способна влиять как на эффективность взаимодействия с аппаратом трансляции в целом, так и на выбор сайтов инициации трансляции. Для исследования транслируемой фракции транскриптома были разработаны экспериментальные методы анализа, наиболее информативным из которых является рибосомное профилирование (РП, Ribo-seq). Первоначально созданный для использования в дрожжевых системах, этот метод был адаптирован для трансляционных исследований на многих видах растений. Технология включает выделение полисомной фракции и высокопроизводительное секвенирование пула сегментов мРНК, связанных с рибосомами. Сравнение результатов покрытия транскриптома прочтениями, полученными по протоколу рибосомного профилирования, с аналогичным результатами по секвенированию транскриптома дает возможность оценить эффективность трансляции для каждого транскрипта. Точные положения рибосом, определенные на последовательностях мРНК, позволяют определять трансляцию открытых рамок считывания и переключение между трансляцией нескольких рамок считывания – феномен, при котором с одной матрицы РНК происходят считывание двух или более перекрывающихся рамок и биосинтез разных белков. Преимущество метода заключается в том, что он дает возможность получить количественные оценки покрытия рибосомами мРНК и может выявлять относительно редкие события трансляции. Использование этой технологии позволило классифицировать гены растений по типу регуляции их экспрессии на уровне транскрипции, трансляции или на обоих уровнях. Обнаружены особенности структуры мРНК, которые влияют на уровни трансляции: формирование квадруплексов G2 и наличие специфических мотивов в области 5’-UTR, GC-состав, наличие альтернативных стартов трансляции, влияние uORF на трансляцию нижестоящих mORF. Показано, что изменения регуляции экспрессии генов на уровне трансляции возникают в ответ на биотический и абиотический стрессы, а также в процессе развития растений. В обзоре кратко рассмотрены методология РП и перспективы ее применения для исследования структурно-функциональной организации и регуляции экспрессии генов растений.

Circular RNA-encoded oncogenic E-cadherin variant promotes glioblastoma tumorigenicity through activation of EGFR-STAT3 signalling.

Activated EGFR signalling drives tumorigenicity in 50% of glioblastoma (GBM). However, EGFR-targeting therapy has proven ineffective in treating patients with GBM, indicating that there is redundant EGFR activation. Circular RNAs are covalently closed RNA transcripts that are involved in various physiological and pathological processes. Herein, we report an additional activation mechanism of EGFR signalling in GBM by an undescribed secretory E-cadherin protein variant (C-E-Cad) encoded by a circular E-cadherin (circ-E-Cad) RNA through multiple-round open reading frame translation. C-E-Cad is overexpressed in GBM and promotes glioma stem cell tumorigenicity. C-E-Cad activates EGFR independent of EGF through association with the EGFR CR2 domain using a unique 14-amino-acid carboxy terminus, thereby maintaining glioma stem cell tumorigenicity. Notably, inhibition of C-E-Cad markedly enhances the antitumour activity of therapeutic anti-EGFR strategies in GBM. Our results uncover a critical role of C-E-Cad in stimulating EGFR signalling and provide a promising approach for treating EGFR-driven GBM.

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.

Conservation of gene architecture and domains amidst sequence divergence in the hsrω lncRNA gene across the Drosophila genus: an in silico analysis

The developmentally active and cell-stress responsive hsrω locus in Drosophila melanogaster carries two exons, one omega intron, one short translatable open reading frame (ORFω), long stretch of unique tandem repeats and an overlapping mir-4951 near its 30' end. It produces multiple long noncoding RNAs (lncRNAs) using two transcription start and four termination sites. Earlier cytogenetic studies revealed functional conservation of hsrω in several Drosophila species. However, sequence analysis in three species showed poor conservation for ORFω, tandem repeat and other regions while the 16 nt at 50 and 60 nt at 30 splice junctions of the omega intron, respectively, were found to be ultra-conserved. The present bioinformatic study using the splice-junction landmarks in D. melanogaster hsrω identified orthologues in publicly available 34 Drosophila species genomes. Each orthologue carries a short ORFω, ultra-conserved splice junctions of omega intron, repeat region, conserved 30'end located at mir-4951, and syntenic neighbours. Multiple copies of conserved nonamer motifs are seen in the tandem repeat region, despite a high variability in the repeat sequences. Intriguingly, only the omega intron sequences in different species show evolutionary relationships matching the general phylogenetic history in the genus. Search in other known insect genomes did not reveal sequence homology although a locus with similar functional properties is suggested in Chironomus and Ceratitis genera. Amidst the high sequence divergence, the conserved organization of exons, ORFω and omega intron in this gene's proximal part and tandem repeats in distal part across the Drosophila genus is remarkable and possibly reflects functional importance of higher order structure of hsrω lncRNAs and the small omega peptide.

Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression.

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5'-untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High-level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.