Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3′ untranslated regions

Ribosome profiling identifies ribosome positions on translated mRNAs. A prominent feature of published datasets is the near complete absence of ribosomes in 3′ untranslated regions (3′UTR) although substantial ribosome density can be observed on non-coding RNAs. Here we perform ribosome profiling in cultured Drosophila and human cells and show that different features of translation are revealed depending on the nuclease and the digestion conditions used. Most importantly, we observe high abundance of ribosome protected fragments in 3′UTRs of thousands of genes without manipulation of translation termination. Affinity purification of ribosomes indicates that the 3′UTR reads originate from ribosome protected fragments. Association of ribosomes with the 3′UTR may be due to ribosome migration through the stop codon or 3′UTR mRNA binding to ribosomes on the coding sequence. This association depends primarily on the relative length of the 3′UTR and may be related to translational regulation or ribosome recycling, for which the efficiency is known to inversely correlate with 3′UTR length. Together our results indicate that ribosome profiling is highly dependent on digestion conditions and that ribosomes commonly associate with the 3′UTR, which may have a role in translational regulation.     

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.     

Conservation and Losses of Non-Coding RNAs in Avian Genomes

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous “losses” of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.     

Evaluating the Effect of 3′-UTR Variants in DICER1 and DROSHA on Their Tissue-Specific Expression by miRNA Target Prediction

Untranslated gene regions (UTRs) play an important role in controlling gene expression. 3′-UTRs are primarily targeted by microRNA (miRNA) molecules that form complex gene regulatory networks. Cancer genomes are replete with non-coding mutations, many of which are connected to changes in tumor gene expression that accompany the development of cancer and are associated with resistance to therapy. Therefore, variants that occurred in 3′-UTR under cancer progression should be analysed to predict their phenotypic effect on gene expression, e.g., by evaluating their impact on miRNA target sites. Here, we analyze 3′-UTR variants in DICER1 and DROSHA genes in the context of myelodysplastic syndrome (MDS) development. The key features of this analysis include an assessment of both “canonical” and “non-canonical” types of mRNA-miRNA binding and tissue-specific profiling of miRNA interactions with wild-type and mutated genes. As a result, we obtained a list of DICER1 and DROSHA variants likely altering the miRNA sites and, therefore, potentially leading to the observed tissue-specific gene downregulation. All identified variants have low population frequency consistent with their potential association with pathology progression.     

Bioinformatics Analysis Identify Novel OB Fold Protein Coding Genes in C. elegans

Background

The C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB) fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5–25%). Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n = 46) compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n = 344) or fruit fly D. melanogaster (n = 84). Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies.

Methodology/Principal Findings

This study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans.

Conclusions/Significance

This study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.     

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs.     

Higher Landing Accuracy in Expert Pilots is Associated with Lower Activity in the Caudate Nucleus

The most common lethal accidents in General Aviation are caused by improperly executed landing approaches in which a pilot descends below the minimum safe altitude without proper visual references. To understand how expertise might reduce such erroneous decision-making, we examined relevant neural processes in pilots performing a simulated landing approach inside a functional MRI scanner. Pilots (aged 20–66) were asked to “fly” a series of simulated “cockpit view” instrument landing scenarios in an MRI scanner. The scenarios were either high risk (heavy fog–legally unsafe to land) or low risk (medium fog–legally safe to land). Pilots with one of two levels of expertise participated: Moderate Expertise (Instrument Flight Rules pilots, n = 8) or High Expertise (Certified Instrument Flight Instructors or Air-Transport Pilots, n = 12). High Expertise pilots were more accurate than Moderate Expertise pilots in making a “land” versus “do not land” decision (CFII: d′ = 3.62±2.52; IFR: d′ = 0.98±1.04; p<.01). Brain activity in bilateral caudate nucleus was examined for main effects of expertise during a “land” versus “do not land” decision with the no-decision control condition modeled as baseline. In making landing decisions, High Expertise pilots showed lower activation in the bilateral caudate nucleus (0.97±0.80) compared to Moderate Expertise pilots (1.91±1.16) (p<.05). These findings provide evidence for increased “neural efficiency” in High Expertise pilots relative to Moderate Expertise pilots. During an instrument approach the pilot is engaged in detailed examination of flight instruments while monitoring certain visual references for making landing decisions. The caudate nucleus regulates saccade eye control of gaze, the brain area where the “expertise” effect was observed. These data provide evidence that performing “real world” aviation tasks in an fMRI provide objective data regarding the relative expertise of pilots and brain regions involved in it.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

Nucleotide Composition of Cellular Internal Ribosome Entry Sites Defines Dependence on NF45 and Predicts a Posttranscriptional Mitotic Regulon

The vast majority of cellular mRNAs initiate their translations through a well-defined mechanism of ribosome recruitment that occurs at the 5′-terminal 7-methylguanosine cap with the help of several canonical protein factors. A subset of cellular and viral mRNAs contain regulatory motifs in their 5′ untranslated regions (UTRs), termed internal ribosome entry sites (IRES), that sidestep this canonical mode of initiation. On cellular mRNAs, this mechanism requires IRES trans-acting protein factors (ITAFs) that facilitate ribosome recruitment downstream of the cap. While several ITAFs and their target mRNAs have been empirically identified, the in silico prediction of targets has proved difficult. Here, we report that a high AU content (>60%) of the IRES-containing 5′ UTRs serves as an excellent predictor of dependence on NF45, a recently identified ITAF. Moreover, we provide evidence that cells deficient in NF45 ITAF activity exhibit reduced IRES-mediated translation of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 (cIAP1) mRNAs that, in turn, leads to dysregulated expression of their respective targets, survivin and cyclin E. This specific defect in IRES translation explains in part the cytokinesis impairment and senescence-like phenotype observed in HeLa cells expressing NF45 RNA interference (RNAi). This study uncovers a novel role for NF45 in regulating ploidy and highlights the importance of IRES-mediated translation in cellular homeostasis.     

Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA

The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5′ and 3′ UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30–40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5′ UTR, while de novo initiation including 5′ UTR scanning proceeds at a much slower rate. Removal or replacements of 5′ and 3′ UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.