Untranslated Region Structure Research Articles

Dysregulated mRNA Translation in the G2019S LRRK2 and LRRK2 Knock-Out Mouse Brains.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) causes familial Parkinson's disease (PD) and is also found in a subset of idiopathic cases. Prior studies in Drosophila and human induced pluripotent stem cell (iPSC)-derived dopamine neurons uncovered a pronounced effect of G2019S LRRK2 on mRNA translation. It was previously reported that G2019S LRRK2 promotes translation of mRNAs with complex 5' untranslated region (UTR) secondary structure, resulting in increased expression of calcium channels and dysregulated calcium homeostasis in human dopamine neurons. Here, we show that dysregulated translation occurs in the brains of mammalian LRRK2 models in vivo Through ribosome profiling studies of global translation, we observe that mRNAs with complex 5'UTR structure are also preferentially translated in the G2019S LRRK2-expressing mouse brain. Reporter assays suggest that this 5'UTR preference is independent of translation initiation factors. Conversely, translation of mRNAs with complex 5'UTR secondary structure is downregulated in LRRK2 knock-out (KO) mouse brain, indicating a robust link between LRRK2 kinase activity and translation of mRNA with complex 5'UTR structure. Further, substantia nigra pars compacta (SNpc) dopamine neurons in the G2019S LRRK2-expressing brain exhibit increased calcium influx, which is consistent with the previous report from human dopamine neurons. These results collectively suggest that LRRK2 plays a mechanistic role in translational regulation, and the G2019S mutation in LRRK2 causes translational defects leading to calcium dysregulation in the mammalian brain.

Genome-Scale Analysis of Acetobacterium woodii Identifies Translational Regulation of Acetogenesis.

ABSTRACTAcetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity.IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

Targeting structural features of viral genomes with a nano-sized supramolecular drug.

RNA targeting is an exciting frontier for drug design. Intriguing targets include functional RNA structures in structurally-conserved untranslated regions (UTRs) of many lethal viruses. However, computational docking screens, valuable in protein structure targeting, fail for inherently flexible RNA. Herein we harness MD simulations with Markov state modeling to enable nanosize metallo-supramolecular cylinders to explore the dynamic RNA conformational landscape of HIV-1 TAR untranslated region RNA (representative for many viruses) replicating experimental observations. These cylinders are exciting as they have unprecedented nucleic acid binding and are the first supramolecular helicates shown to have anti-viral activity in cellulo: the approach developed in this study provides additional new insight about how such viral UTR structures might be targeted with the cylinder binding into the heart of an RNA-bulge cavity, how that reduces the conformational flexibility of the RNA and molecular details of the insertion mechanism. The approach and understanding developed represents a new roadmap for design of supramolecular drugs to target RNA structural motifs across biology and nucleic acid nanoscience.

The RNA Architecture of the SARS-CoV-2 3'-Untranslated Region.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.

Disruption of Zika Virus xrRNA1-Dependent sfRNA1 Production Results in Tissue-Specific Attenuated Viral Replication.

The Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths that result from the exonuclease degradation of the viral 3′ untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of an incomplete viral genome degradation of the 3′ UTR by the host 5′ to 3′ exoribonuclease, Xrn1. The halting of Xrn1 in the 3′ UTR is due to two RNA pseudoknot structures in the 3′ UTR, termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we generate a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3′ UTR structure. We find that the X1 virus lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult Ifnar1−/− mice, we find that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to wild-type ZIKV infection. Despite the attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that the targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.

The cellular stress response and temperature: Function, regulation, and evolution.

The cellular stress response (CSR) is critical for enabling organisms to cope with thermal damage to proteins, nucleic acids, and membranes. It is a graded response whose properties vary with the degree of cellular damage. Molecular damage has positive, as well as negative, function-perturbing effects. Positive effects include crucial regulatory interactions that orchestrate involvement of the different components of the CSR. Thermally unfolded proteins signal for rapid initiation of transcription of genes encoding heat shock proteins (HSPs), central elements of the heat shock response (HSR). Thermal disruption of messenger RNA (mRNA) secondary structures in untranslated regions leads to the culling of the mRNA pool: thermally labile mRNAs for housekeeping proteins are degraded by exonucleases; heat-resistant mRNAs for stress proteins like HSPs then can monopolize the translational apparatus. Thus, proteins and RNA function as "cellular thermometers," and evolved differences in their thermal stabilities enable rapid initiation of the CSR whenever cell temperature rises significantly above the normal thermal range of a species. Covalent DNA damage, which may result from increased production of reactive oxygen species, is temperature-dependent; its extent may determine cellular survival. High levels of stress that exceed capacities for molecular repair can lead to proteolysis, inhibition of cell division, and programmed cell death (apoptosis). Onset of these processes may occur later in the stress period, after initiation of the HSR, to allow HSPs opportunity to restore protein homeostasis. Delay of these energy costly processes may also result from shortfalls in availability of adenosine triphosphate and reducing power during times of peak stress.

Human astroviruses: in silico analysis of the untranslated region and putative binding sites of cellular proteins.

Human astrovirus (HAstV) constitutes a major cause of acute gastroenteritis in children. The viral 5′ and 3′ untranslated regions (UTR) have been involved in the regulation of several molecular mechanisms. However, in astrovirues have been less characterized. Here, we analyzed the secondary structures of the 5′ and 3′ UTR of HAstV, as well as their putative target sites that might be recognized by cellular factors. To our knowledge, this is the first bioinformatic analysis that predicts the HAstV 5′ UTR secondary structure. The analysis showed that both the UTR sequence and secondary structure are highly conserved in all HAstVs analyzed, suggesting their regulatory role of viral activities. Notably, the UTRs of HAstVs contain putative binding sites for the serine/arginine-rich factors SRSF2, SRSF5, SRSF6, SRSF3, and the multifunctional hnRNPE2 protein. More importantly, putative binding sites for PTB were localized in single-stranded RNA sequences, while hnRNPE2 sites were localized in double-stranded sequence of the HAstV 5′ and 3′ UTR structures. These analyses suggest that the combination of SRSF proteins, hnRNPE2 and PTB described here could be involved in the maintenance of the secondary structure of the HAstVs, possibly allowing the recruitment of the replication complex that selects and recruits viral RNA replication templates.Electronic supplementary materialThe online version of this article (10.1007/s11033-018-4498-8) contains supplementary material, which is available to authorized users.

Analyses of mRNA structure dynamics identify embryonic gene regulatory programs.

RNA folding plays a crucial role in RNA function. However, knowledge of the global structure of the transcriptome is limited to cellular systems at steady state, thus hindering the understanding of RNA structure dynamics during biological transitions and how it influences gene function. Here, we characterized mRNA structure dynamics during zebrafish development. We observed that on a global level, translation guides structure rather than structure guiding translation. We detected a decrease in structure in translated regions and identified the ribosome as a major remodeler of RNA structure in vivo. In contrast, we found that 3' untranslated regions (UTRs) form highly folded structures in vivo, which can affect gene expression by modulating microRNA activity. Furthermore, dynamic 3'-UTR structures contain RNA-decay elements, such as the regulatory elements in nanog and ccna1, two genes encoding key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.

MiR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5' UTR RNA to stabilize and promote viral RNA accumulation.

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5′ UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5′ untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5′ UTR RNA folding, and mutant 5′ UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5′ UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5′ end annealing or 3′ overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5′ UTR, in conjunction with any Ago isoform, modifies the 5′ UTR structure to stabilize the viral genome and promote HCV RNA accumulation.

Synthetic Riboswitches: From Plug and Pray toward Plug and Play.

In synthetic biology, metabolic engineering, and gene therapy, there is a strong demand for orthogonal or externally controlled regulation of gene expression. Here, RNA-based regulatory devices represent a promising emerging alternative to proteins, allowing a fast and direct control of gene expression, as no synthesis of regulatory proteins is required. Besides programmable ribozyme elements controlling mRNA stability, regulatory RNA structures in untranslated regions are highly interesting for engineering approaches. Riboswitches are especially well suited, as they show a modular composition of sensor and response elements, allowing a free combination of different modules in a plug-and-play-like mode. The sensor or aptamer domain specifically interacts with a trigger molecule as a ligand, modulating the activity of the adjacent response domain that controls the expression of the genes located downstream, in most cases at the level of transcription or translation. In this review, we discuss the recent advances and strategies for designing such synthetic riboswitches based on natural or artificial components and readout systems, from trial-and-error approaches to rational design strategies. As the past several years have shown dramatic development in this fascinating field of research, we can give only a limited overview of the basic riboswitch design principles that is far from complete, and we apologize for not being able to consider every successful and interesting approach described in the literature.

Identification and genetic characterization of Zika virus isolated from an imported case in China

Zika virus (ZIKV) is a reemerging flavivirus that stroke Brazil in 2015 and appeared in China for the first time in 2016. Sequencing and genomic analysis are essential for Zika virus study. However, the complete genome length of Zika virus is still a disputable issue. In this study, we reported the complete genomic sequence of Zika virus strain ZKC2/2016 from an imported case in China, in February 2016. The virus was isolated and virus characteristics were identified by cytopathic effect, quantitative real time-PCR, immunofluorescence assay and electronic microscopy. Next generation sequencing (NGS) technique and 5′ and 3′ Rapid Amplification cDNA Ends (RACE) PCR were used to sequence the complete genome. The genome length of this newly obtained Zika virus strain is 10,807base pairs (bp). Genetic analysis showed that ZKC2/2016, along with other Chinese Zika virus strains in 2016, formed three clusters within Asian linage. Multiple sequence alignment and prediction of RNA secondary structure of untranslated regions (UTRs) of ZKC2/2016 and several other ZIKV strains indicated that the difference of ZIKV genome length mainly laid in UTRs. Besides, those genomes shorter than 10,790bp were probably incomplete due to lacking conserved secondary RNA structures in untranslated regions which were playing important roles in flavivirus replication. Our findings benefited the disease control of Zika fever in China and the study of Zika virus genome.

Whole-Genome Sequencing Analysis from the Chikungunya Virus Caribbean Outbreak Reveals Novel Evolutionary Genomic Elements.

BackgroundChikungunya virus (CHIKV), an alphavirus and member of the Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naïve populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak.Methodology/Principle FindingsWe used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3’ untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells.Conclusions/SignificanceHere we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3’UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naïve population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV.

IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance.

Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3′ untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3′ UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217 G allele mRNA forms well-defined 3′ UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure.

Transcripts with in silico predicted RNA structure are enriched everywhere in the mouse brain

BackgroundPost-transcriptional control of gene expression is mostly conducted by specific elements in untranslated regions (UTRs) of mRNAs, in collaboration with specific binding proteins and RNAs. In several well characterized cases, these RNA elements are known to form stable secondary structures. RNA secondary structures also may have major functional implications for long noncoding RNAs (lncRNAs). Recent transcriptional data has indicated the importance of lncRNAs in brain development and function. However, no methodical efforts to investigate this have been undertaken. Here, we aim to systematically analyze the potential for RNA structure in brain-expressed transcripts.ResultsBy comprehensive spatial expression analysis of the adult mouse in situ hybridization data of the Allen Mouse Brain Atlas, we show that transcripts (coding as well as non-coding) associated with in silico predicted structured probes are highly and significantly enriched in almost all analyzed brain regions. Functional implications of these RNA structures and their role in the brain are discussed in detail along with specific examples. We observe that mRNAs with a structure prediction in their UTRs are enriched for binding, transport and localization gene ontology categories. In addition, after manual examination we observe agreement between RNA binding protein interaction sites near the 3’ UTR structures and correlated expression patterns.ConclusionsOur results show a potential use for RNA structures in expressed coding as well as noncoding transcripts in the adult mouse brain, and describe the role of structured RNAs in the context of intracellular signaling pathways and regulatory networks. Based on this data we hypothesize that RNA structure is widely involved in transcriptional and translational regulatory mechanisms in the brain and ultimately plays a role in brain function.

Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3’ UTR structures

Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines. The DENV-2 positive-strand RNA genome contains 5’ and 3’ untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins. In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5’ and 3’ UTRs as “bait”, combined with quantitative mass spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs. Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3’ UTR, possibly by formation of a pseudoknot structure. Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3’ UTR. These results suggest that the DENV-2 3’ UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3’ UTR is required for DENV replication.

Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally through binding specific sites within the 3' untranslated regions (UTRs) of their target mRNAs. Numerous investigations have documented repressive effects of miRNAs and identified factors required for their activity. However, the precise mechanisms by which miRNAs modulate gene expression are still obscure. Here, we have examined the effects of multiple miRNAs on diverse target transcripts containing artificial or naturally occurring 3' UTRs in human cell culture. In agreement with previous studies, we report that both the 5' m(7)G cap and 3' poly(A) tail are essential for maximum miRNA repression. These cis-acting elements also conferred miRNA susceptibility to target mRNAs translating under the control of viral- and eukaryotic mRNA-derived 5' UTR structures that enable cap-independent translation. Additionally, we evaluated a role for the poly(A)-binding protein (PABP) in miRNA function utilizing multiple approaches to modulate levels of active PABP in cells. PABP expression and activity inversely correlated with the strength of miRNA silencing, in part due to antagonism of target mRNA deadenylation. Together, these findings further define the cis- and trans-acting factors that modulate miRNA efficacy.

Structural and functional analyses of the 3′ untranslated region of Bamboo mosaic virus satellite RNA

The 3′-untranslated region (UTR) of RNA genomes of viruses and satellite RNAs plays essential roles in viral replication and transcription. The structural features of the 3′-UTR of the satellite RNA of Bamboo mosaic virus (satBaMV) involved in its replication were analyzed in this study. By the use of enzymatic probing, the secondary structure of satBaMV 3′-UTR was confirmed to comprise two small stem-loops (SLA and SLB), one large stem-loop (SLC), and a poly(A) tail of mainly 75–200 adenylate residues, which is similar to those on the genomic RNA of the helper virus, BaMV. Five sets of mutants of satBaMV were constructed to analyze the biological functions of the structural elements of the 3′-UTR. The data revealed that both the polyadenylation signal and poly(A) tail are required for satBaMV RNA replication. The structural conservation of SLA, SLB, and SLC is also important for efficient satBaMV accumulation, whereas the nucleotides in these regions may also possess sequence-specific functions. In contrast to the requirement for the accumulation of BaMV genomic RNA, mutations in the conserved hexanucleotide (ACCUAA) in the loop region of SLC had limited effect on the accumulation of satBaMV RNA. In addition, replacing the 5′-, 3′-UTR, or both regions of satBaMV by those of BaMV greatly decreased the accumulation of satBaMV RNA. Taken together, these data indicate that satBaMV might have adopted a 3′-UTR structure similar to that of BaMV but may have evolved distinct features for its efficient replication.

Genetic Code Supports Targeted Insertion of Two Amino Acids by One Codon

Strict one-to-one correspondence between codons and amino acids is thought to be an essential feature of the genetic code. However, we report that one codon can code for two different amino acids with the choice of the inserted amino acid determined by a specific 3' untranslated region structure and location of the dual-function codon within the messenger RNA (mRNA). We found that the codon UGA specifies insertion of selenocysteine and cysteine in the ciliate Euplotes crassus, that the dual use of this codon can occur even within the same gene, and that the structural arrangements of Euplotes mRNA preserve location-dependent dual function of UGA when expressed in mammalian cells. Thus, the genetic code supports the use of one codon to code for multiple amino acids.

C. elegans and H. sapiens mRNAs with edited 3′ UTRs are present on polysomes

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive double-stranded 3' UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 3' UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 3' UTRs are present on translating ribosomes.

Genetic Adaptation to Untranslated Region-Mediated Enterovirus Growth Deficits by Mutations in the Nonstructural Proteins 3AB and 3CD

Both untranslated regions (UTRs) of plus-strand RNA virus genomes jointly control translation and replication of viral genomes. In the case of the Enterovirus genus of the Picornaviridae family, the 5'UTR consists of a cloverleaf-like terminus preceding the internal ribosomal entry site (IRES) and the 3' terminus is composed of a structured 3'UTR and poly(A). The IRES and poly(A) have been implicated in translation control, and all UTR structures, in addition to cis-acting genetic elements mapping to the open reading frame, have been assigned roles in RNA replication. Viral UTRs are recognized by viral and host cell RNA-binding proteins that may co-determine genome stability, translation, plus- and minus-strand RNA replication, and scaffolding of viral replication complexes within host cell substructures. In this report, we describe experiments with coxsackie B viruses with a cell type-specific propagation deficit in Sk-N-Mc neuroblastoma cells conferred by the combination of a heterologous IRES and altered 3'UTR. Serial passage of these constructs in Sk-N-Mc cells yielded genetic adaptation by mutations within the viral nonstructural proteins 3A and 3C. Our data implicate 3A and/or 3C or their precursors 3AB and/or 3CD in a functional complex with the IRES and 3'UTR that drives viral propagation. Adaptation to neuroblastoma cells suggests an involvement of cell type-specific host factors or the host cell cytoplasmic milieu in this phenomenon.