Predicting functional upstream open reading frames in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Some upstream open reading frames (uORFs) regulate gene expression (i.e., they are functional) and can play key roles in keeping organisms healthy. However, how uORFs are involved in gene regulation is not yet fully understood. In order to get a complete view of how uORFs are involved in gene regulation, it is expected that a large number of experimentally verified functional uORFs are needed. Unfortunately, wet-experiments to verify that uORFs are functional are expensive.</p> <p>Results</p> <p>In this paper, a new computational approach to predicting functional uORFs in the yeast <it>Saccharomyces cerevisiae </it>is presented. Our approach is based on inductive logic programming and makes use of a novel combination of knowledge about biological conservation, Gene Ontology annotations and genes' responses to different conditions. Our method results in a set of simple and informative hypotheses with an estimated sensitivity of 76%. The hypotheses predict 301 further genes to have 398 novel functional uORFs. Three (<it>RPC11</it>, <it>TPK1</it>, and <it>FOL1</it>) of these 301 genes have been hypothesised, following wet-experiments, by a related study to have functional uORFs. A comparison with another related study suggests that eleven of the predicted functional uORFs from genes <it>LDB17</it>, <it>HEM3</it>, <it>CIN8</it>, <it>BCK2</it>, <it>PMC1</it>, <it>FAS1</it>, <it>APP1</it>, <it>ACC1</it>, <it>CKA2</it>, <it>SUR1</it>, and <it>ATH1 </it>are strong candidates for wet-lab experimental studies.</p> <p>Conclusions</p> <p>Learning based prediction of functional uORFs can be done with a high sensitivity. The predictions made in this study can serve as a list of candidates for subsequent wet-lab verification and might help to elucidate the regulatory roles of uORFs.</p

Conserved upstream open reading frames in higher plants

Background Upstream open reading frames (uORFs) can down-regulate the translation of the main open reading frame (mORF) through two broad mechanisms: ribosomal stalling and reducing reinitiation efficiency. In distantly related plants, such as rice and Arabidopsis, it has been found that conserved uORFs are rare in these transcriptomes with approximately 100 loci. It is unclear how prevalent conserved uORFs are in closely related plants. Results We used a homology-based approach to identify conserved uORFs in five cereals (monocots) that could potentially regulate translation. Our approach used a modified reciprocal best hit method to identify putative orthologous sequences that were then analysed by a comparative R-nomics program called uORFSCAN to find conserved uORFs. Conclusion This research identified new genes that may be controlled at the level of translation by conserved uORFs. We report that conserved uORFs are rare (&lt;150 loci contain them) in cereal transcriptomes, are generally short (less than 100 nt), highly conserved (50% median amino acid sequence similarity), position independent in their 5'-UTRs, and their start codon context and the usage of rare codons for translation does not appear to be important.Michael K Tran, Carolyn J Schultz and Ute Bauman

New peptides under the s(ORF)ace of the genome

Hundreds of previously unidentified functional small peptides could exist in most genomes, but these sequences have been generally overlooked. The discovery of genes encoding small peptides with important functions in different organisms, has ignited the interest in these sequences, and led to an increasing amount of effort towards their identification. Here, we review the advances, both, computational, and biochemical, that are leading the way in the discovery of putatively functional smORFs, as well as the functional studies that have been carried out as a consequence of these searches. The evidence suggests that smORFs form a substantial part of our genomes, and that their encoded peptides could have important functions in a variety of cellular function

The Role of Upstream Open Reading Frames in Regulating Neuronal Protein Synthesis

Spatial and temporal control of protein synthesis in response to activity is required for neuronal function and plasticity. mRNA structure and sequence provide a powerful platform for such regulation, but how such information is utilized in neurons is incompletely understood. In my thesis, I explore how functional elements within 5’leaders (traditionally termed 5’UTR or untranslated region) of mRNAs act as cis-regulatory elements to influence basal and activity-dependent translation in neurons. First, I identified a specific role for upstream open reading frames (uORFs) in regulating mRNA translation during neuronal differentiation. uORFs are regions within the 5’ leader that undergo translation. Using ribosome profiling (RP), an emerging next-generation sequencing technique which utilizes a modified RNA-sequencing library preparation to detect regions of mRNA occupied by actively translating ribosomes, I identified thousands of uORFs in human neuroblastoma cells. A portion of these uORFs demonstrated clear usage shifts with differentiation. Highly conserved uORFs exhibited increased GC content and were associated with cumulatively repressed CDSs. Importantly, changes in the translational efficiency of these conserved uORFs across differentiation were inversely correlated with CDS translation on these same transcripts. These data demonstrate uORF usage is common in neuroblastoma cells and that specific uORFs act as regulators of cell state-specific translation in neuronal differentiation. Next, I investigated the function of CGG repeats in the 5’ leader of FMR1. All humans have a conserved CGG-trinucleotide repeat (typically 20-45 repeats) in FMR1 that can become unstable and expand intergenerationally. Large expansions (>200 CGG repeats) cause Fragile X Syndrome, a common cause of intellectual disability, by silencing FMR1, leading to loss of the fragile X protein, FMRP. Intermediate (55-200 CGGs) expansions, in contrast, are transcribed and cause an age-related neurodegenerative condition known as Fragile-X Associated Tremor/Ataxia Syndrome (FXTAS). Our lab discovered that this repeat facilitates Repeat Associated Non-AUG translation (RANT), whereby ribosomes initiate at non-AUG codons upstream of the repeat to produce toxic homopolymeric proteins that drive pathogenesis in FXTAS. FMR1 avidly supports RANT at normal repeat sizes, suggesting that it might serve as a regulatory uORF to control FMRP synthesis. To address this, I expressed nanoluciferase reporters in rat hippocampal neurons. Using this strategy, I found that RANT exhibits a strong negative effect on FMRP synthesis at both normal and expanded repeats. FMRP is a key synaptic protein that is rapidly synthesized in response to mGluR activity. Importantly, preventing RANT or removing the repeat itself blocked this mGluR-induced response. This suggests that FMR1 relies on these two elements to appropriately scale synaptic FMRP synthesis. Using non-cleaving antisense oligonucleotides (ASOs) that target the RANT initiation sites, I found that blocking RANT could decrease toxic protein production and prevent neuronal death. In a line of iPSC-derived neurons from a patient with a large CGG repeat (>200) that still generates FMR1 mRNA but has deficits in FMRP, treatment with the ASO increased endogenous FMRP expression by 50%. These findings define a native function for RANT and CGG repeats in regulating FMRP synthesis, and delineate RANT as a therapeutic target in Fragile X-associated disorders.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143943/1/ctln_1.pd

Novel targets of eiF2 kinases determine cell fate during the integrated stress response

Indiana University-Purdue University Indianapolis (IUPUI)Eukaryotic cells rapidly modulate protein synthesis in response to environmental cues through the reversible phosphorylation of eukaryotic initiation factor 2 (eIF2α~P) by a family of eIF2α kinases. The eIF2 delivers initiator Met-tRNAiMet to the translational apparatus, and eIF2α~P transforms its function from a translation initiation factor into a competitive inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which is responsible for the recycling of eIF2-GDP to the translationally-competent eIF2-GTP state. Reduced eIF2-GTP levels lower general protein synthesis, which allows for the conservation of energy and nutrients, and a restructuring of gene expression. Coincident with global translational control, eIF2α~P directs the preferential translation of mRNA encoding ATF4, a transcriptional activator of genes important for stress remediation. The term Integrated Stress Response (ISR) describes this pathway in which multiple stresses converge to phosphorylate eIF2α and enhance synthesis of ATF4 and its downstream effectors. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKα as being subject to both translational and transcriptional induction during eIF2α~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves the stress-induced relief of two inhibitory uORFs in the 5’-leader of the transcript. Also identified as being subject to preferential translation is mRNA encoding the bifunctional aminoacyl tRNA synthetase EPRS. During eIF2α~P, translational regulation of EPRS is suggested to occur through the bypass of a non-canonical upstream ORF encoded by a CUG start codon, highlighting the diversity by which upstream translation initiation events can regulate expression of a downstream coding sequence. This body of work provides for a better understanding of how translational control during stress is modulated genome-wide and for the processes by which this mode of gene regulation in the ISR contributes to cell fate

Development and Application of Next-Generation Sequencing Methods to Profile Cellular Translational Dynamics

The transmission of genetic information from the transcription of DNA to RNA and the subsequent translation of RNA into protein is often abstracted into a linear process. However, as methods and technologies to measure the genomic, transcriptomic, and proteomic content of cells have advanced, so too has our understanding that the transmission of genetic information does not always flow in a lossless manner. For instance, changes observed in messenger RNA (mRNA) abundance are not always retained at the proteomic level. Indeed, a diverse array of mechanisms have been identified that exert regulatory control over this transmission of information. Next-generation short read sequencing has driven many of these insights and provided increasingly nuanced understanding of these regulatory mechanisms. However, the continued development and application of sequencing methodologies and analytics are required to properly contextualize many of these insights on a more global scale. Ribosome profiling is one such recent advancement which enriches for ribosome-protected fragments of mRNA; sequencing and analysis of these ribosome-protected mRNA fragments enables profiling of the translational content of a sample. The aim of this dissertation is to address the need for the development and application of statistical and analytical algorithms to profile the regulatory factors that contribute to the translational dynamics in cells. In the first chapter, I survey the development and application of next-generation sequencing methods for the profiling and computational analysis of translation and translational dynamics. In the second chapter of this thesis, I present SPECtre, a software package that identifies regions of active translation through measurement of the translational engagement of ribosomes over a transcript. SPECtre achieves high sensitivity and specificity in its classification of regions undergoing translation by leveraging the codon-dependent elongation of peptides; this tri-nucleotide periodicity is evident in the alignment of ribosome profiling sequence reads to a reference transcriptome. SPECtre classifies actively translated transcripts according to their coherence in read coverage over a region to an optimal tri-nucleotide signal. In the third chapter, I describe the application of SPECtre to identify the translation of upstream-initiated open-reading frames that may regulate differentiation in a neuron-like cell model. uORFs are transcripts that result from the initiation of translation from AUG, and under certain biological constraints, from non-AUG sequences localized in the 5’ untranslated regions of annotated protein-coding genes. Subsets of these uORFs have been implicated in the regulation of their downstream protein-coding genes in yeast, mice and humans. In this chapter, I provide further evidence for this regulation as well as the spatial context for the functional consequences of uORF translation on downstream protein-coding genes in a neuron-like cell line model of differentiation. Finally, in the fourth chapter, I outline a strategy using our coherence-based translational scoring algorithm to profile ribosomal engagement over chimeric gene fusion breakpoints in prostate cancer. Here, known breakpoints from current annotation databases are integrated with novel junctions nominated by existing whole genome and transcriptomic gene fusion detection algorithms, and the translational profile over these chimeric junctions using SPECtre is measured. This provides an additional layer of translational evidence to known and novel gene fusion breakpoints in prostate cancer. Ongoing development of a database and visualization platform based on these results will enable integrative insights into the transcriptional and translational topology of these breakpoints.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144106/1/stonyc_1.pd

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell