Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

High-throughput identification of RNA localization elements in neuronal cells.

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.

Upstream Flanking Sequence Assists Folding of an RNA Thermometer.

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5'UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5'UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.

Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors.

Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base-base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3' end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.

Beyond protein modification: the rise of non-canonical ADP-ribosylation.

ADP-ribosylation has primarily been known as post-translational modification of proteins. As signalling strategy conserved in all domains of life, it modulates substrate activity, localisation, stability or interactions, thereby regulating a variety of cellular processes and microbial pathogenicity. Yet over the last years, there is increasing evidence of non-canonical forms of ADP-ribosylation that are catalysed by certain members of the ADP-ribosyltransferase family and go beyond traditional protein ADP-ribosylation signalling. New macromolecular targets such as nucleic acids and new ADP-ribose derivatives have been established, notably extending the repertoire of ADP-ribosylation signalling. Based on the physiological relevance known so far, non-canonical ADP-ribosylation deserves its recognition next to the traditional protein ADP-ribosylation modification and which we therefore review in the following.

Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA.

Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5' splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and other minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.

Evidence in disease and non-disease contexts that nonsense mutations cause altered splicing via motif disruption.

Transcripts containing premature termination codons (PTCs) can be subject to nonsense-associated alternative splicing (NAS). Two models have been evoked to explain this, scanning and splice motif disruption. The latter postulates that exonic cis motifs, such as exonic splice enhancers (ESEs), are disrupted by nonsense mutations. We employ genome-wide transcriptomic and k-mer enrichment methods to scrutinize this model. First, we show that ESEs are prone to disruptive nonsense mutations owing to their purine richness and paucity of TGA, TAA and TAG. The motif model correctly predicts that NAS rates should be low (we estimate 5-30%) and approximately in line with estimates for the rate at which random point mutations disrupt splicing (8-20%). Further, we find that, as expected, NAS-associated PTCs are predictable from nucleotide-based machine learning approaches to predict splice disruption and, at least for pathogenic variants, are enriched in ESEs. Finally, we find that both in and out of frame mutations to TAA, TGA or TAG are associated with exon skipping. While a higher relative frequency of such skip-inducing mutations in-frame than out of frame lends some credence to the scanning model, these results reinforce the importance of considering splice motif modulation to understand the etiology of PTC-associated disease.

Co-expression analysis identifies networks of miRNAs implicated in biological ageing and modulated by short-term interval training.

Exercise training seems to promote healthy biological ageing partly by inducing telomere maintenance, yet the molecular mechanisms are not fully understood. Recent studies have emphasised the importance of microRNAs (miRNAs) in ageing and their ability to mirror pathophysiological alterations associated with age-related diseases. We examined the association between aerobic fitness and leukocyte telomere length before determining the influence of vigorous exercise training on the regulation of leukocyte miRNA networks. Telomere length was positively correlated to aerobic fitness (r = 0.32, p = 0.02). 104 miRNAs were differentially expressed after six weeks of thrice-weekly sprint interval training (SIT) in healthy men (q < 0.05). Gene co-expression analysis (WGCNA) detected biologically meaningful miRNA networks, five of which were significantly correlated with pre-SIT and post-SIT expression profiles (p < 0.001) and telomere length. Enrichment analysis revealed that the immune response, T cell differentiation and lipid metabolism associated miRNAs clusters were significantly down-regulated after SIT. Using data acquired from the Gene Expression Omnibus (GEO), we also identified two co-expressed miRNAs families that were modulated by exercise training in previous investigations. Collectively, our findings highlight the miRNA networks implicated in exercise adaptations and telomere regulation, and suggest that SIT may attenuate biological ageing through the control of the let-7 and miR-320 miRNA families.

Mathematical Modeling of ceRNA-Based Interactions.

Competing endogenous RNA (ceRNA) molecules have emerged as key players in regulating gene expression, increasing the complexity of the range of possible dynamics within a cell. The actions of competing RNA typically are sponging behaviors, in a manner that fine-tunes gene expression, but there are particular network structures that may show destabilization due to ceRNA interactions. In this chapter, we discuss how these interactions can be modeled and probed from a mathematical, first-principles perspective.

Pseudogenes as Competitive Endogenous RNAs: Target Prediction and Validation.

Pseudogenes may regulate expression of their parental genes as well as other protein-coding genes through various mechanisms. One such mechanism is the ability to act as competitive endogenous RNA (ceRNA) and participate in microRNA-mediated cross-regulation. Here, we outline how to predict the targets of pseudogene ceRNAs bioinformatically and how to validate them experimentally.

Pseudogenes as Competitive Endogenous RNAs: Testing miRNA Dependency.

Pseudogenes may function as competitive endogenous RNAs (ceRNAs), where they regulate the expression of genes by sequestering shared miRNAs. ceRNAs are becoming more extensively identified and studied, and demonstrating the dependence of their effects on miRNA sequestration is critical to establish them as ceRNAs. Here, we outline an experimental approach to assess the miRNA dependency of a candidate pseudogene ceRNA.

An Overview of the Computational Models Dealing with the Regulatory ceRNA Mechanism and ceRNA Deregulation in Cancer.

Pools of RNA molecules can act as competing endogenous RNAs (ceRNAs) and indirectly alter their expression levels by competitively binding shared microRNAs. This ceRNA cross talk yields an additional posttranscriptional regulatory layer, which plays key roles in both physiological and pathological processes. MicroRNAs can act as decoys by binding multiple RNAs, as well as RNAs can act as ceRNAs by competing for binding multiple microRNAs, leading to many cross talk interactions that could favor significant large-scale effects in spite of the weakness of single interactions. Identifying and studying these extended ceRNA interaction networks could provide a global view of the fine-tuning gene regulation in a wide range of biological processes and tumor progressions. In this chapter, we review current progress of predicting ceRNA cross talk, by summarizing the most up-to-date databases, which collect computationally predicted and/or experimentally validated miRNA-target and ceRNA-ceRNA interactions, as well as the widespread computational methods for discovering and modeling possible evidences of ceRNA-ceRNA interaction networks. These methods can be grouped in two categories: statistics-based methods exploit multivariate analysis to build ceRNA networks, by considering the miRNA expression levels when evaluating miRNA sponging relationships; mathematical methods build deterministic or stochastic models to analyze and predict the behavior of ceRNA cross talk.

WEVar: a novel statistical learning framework for predicting noncoding regulatory variants.

Understanding the functional consequence of noncoding variants is of great interest. Though genome-wide association studies or quantitative trait locus analyses have identified variants associated with traits or molecular phenotypes, most of them are located in the noncoding regions, making the identification of causal variants a particular challenge. Existing computational approaches developed for prioritizing noncoding variants produce inconsistent and even conflicting results. To address these challenges, we propose a novel statistical learning framework, which directly integrates the precomputed functional scores from representative scoring methods. It will maximize the usage of integrated methods by automatically learning the relative contribution of each method and produce an ensemble score as the final prediction. The framework consists of two modes. The first 'context-free' mode is trained using curated causal regulatory variants from a wide range of context and is applicable to predict regulatory variants of unknown and diverse context. The second 'context-dependent' mode further improves the prediction when the training and testing variants are from the same context. By evaluating the framework via both simulation and empirical studies, we demonstrate that it outperforms integrated scoring methods and the ensemble score successfully prioritizes experimentally validated regulatory variants in multiple risk loci.

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA.

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Precision of tissue patterning is controlled by dynamical properties of gene regulatory networks.

During development, gene regulatory networks allocate cell fates by partitioning tissues into spatially organised domains of gene expression. How the sharp boundaries that delineate these gene expression patterns arise, despite the stochasticity associated with gene regulation, is poorly understood. We show, in the vertebrate neural tube, using perturbations of coding and regulatory regions, that the structure of the regulatory network contributes to boundary precision. This is achieved, not by reducing noise in individual genes, but by the configuration of the network modulating the ability of stochastic fluctuations to initiate gene expression changes. We use a computational screen to identify network properties that influence boundary precision, revealing two dynamical mechanisms by which small gene circuits attenuate the effect of noise in order to increase patterning precision. These results highlight design principles of gene regulatory networks that produce precise patterns of gene expression.

Massively Parallel Analysis of Regulatory RNA Sequences.

The stability of RNA transcripts is regulated by signals within their sequences, but the identity of those signals still remain elusive in many biological systems. Recently introduced massively parallel tools for the analysis of regulatory RNA sequences provide the ability to detect functional cis-regulatory sequences of post-transcriptional RNA regulation at a much larger scale and resolution than before. Their application formulates the underlying sequence-based rules and predicts the impact of genetic variations. Here, we describe the application of UTR-Seq, as a strategy to uncover cis-regulatory signals of RNA stability during early zebrafish embryogenesis. The method combines massively parallel reporter assays (MPRA) with computational regression models. It surveys the effect of tens of thousands of regulatory sequences on RNA stability and analyzes the results via regression models to identify sequence signals that impact RNA stability and to predict the in vivo effect of sequence variations.

Identification of 3' UTR motifs required for mRNA localization to myelin sheaths in vivo.

Myelin is a specialized membrane produced by oligodendrocytes that insulates and supports axons. Oligodendrocytes extend numerous cellular processes, as projections of the plasma membrane, and simultaneously wrap multiple layers of myelin membrane around target axons. Notably, myelin sheaths originating from the same oligodendrocyte are variable in size, suggesting local mechanisms regulate myelin sheath growth. Purified myelin contains ribosomes and hundreds of mRNAs, supporting a model that mRNA localization and local protein synthesis regulate sheath growth and maturation. However, the mechanisms by which mRNAs are selectively enriched in myelin sheaths are unclear. To investigate how mRNAs are targeted to myelin sheaths, we tested the hypothesis that transcripts are selected for myelin enrichment through consensus sequences in the 3' untranslated region (3' UTR). Using methods to visualize mRNA in living zebrafish larvae, we identified candidate 3' UTRs that were sufficient to localize mRNA to sheaths and enriched near growth zones of nascent membrane. We bioinformatically identified motifs common in 3' UTRs from 3 myelin-enriched transcripts and determined that these motifs are required and sufficient in a context-dependent manner for mRNA transport to myelin sheaths. Finally, we show that 1 motif is highly enriched in the myelin transcriptome, suggesting that this sequence is a global regulator of mRNA localization during developmental myelination.

Multi-omics analysis of copy number variations of RNA regulatory genes in soft tissue sarcoma.

AIMS: RNA regulatory genes were closely associated with tumorigenesis and prognosis in multiple tumors. Copy number variation (CNV) is a frequent characteristic in soft tissue sarcomas (STS). However, little is known regarding their possible roles in STS. MAIN METHODS: RNA sequence profiles and CNV data of 255 STS patients were downloaded from the Cancer Genome Atlas (TCGA). The correlation analysis involved CNVs of RNA regulatory genes, patient survival, immune infiltration, and DNA methylation. Drug sensitivity (IC50) was analyzed and validated by MTT assays in STS cell lines. KEY FINDINGS: CNV events were frequently observed in all kinds (m6A, m5C, ac4C, m1A, m3C, m6Am, m7G, and Ψ) of RNA regulatory genes. Diploid copy number (CN) of METTL4 was associated with better overall survival (OS) in STS and the subtypes (leiomyosarcoma, LMS; dedifferentiated liposarcoma, DDLPS). In STS and LMS, diploid CN of METTL4 was significantly associated with higher infiltration fraction of resting mast cells. In STS and DDLPS, diploid CN of METTL4 possessed lower methylation level in CpG site of cg12105018, which represented better OS. Besides, sensitive drugs for STS cell lines were analyzed according to lower IC50 for the loss CN of METTL4. Temozolomide and Olaparib were identified. Further validation by MTT assays demonstrated that GCT was the most sensitive cell line to both Temozolomide and Olaparib. SIGNIFICANCE: CNV of METTL4 could be a prognostic biomarker for STS by potentially influencing mast cell infiltration and DNA methylation. Besides, STS with loss CN of METTL4 would be sensitive to Temozolomide and Olaparib.

Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome.

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

ACE2 polymorphisms as potential players in COVID-19 outcome.

The clinical condition COVID-19, caused by SARS-CoV-2, was declared a pandemic by the WHO in March 2020. Currently, there are more than 5 million cases worldwide, and the pandemic has increased exponentially in many countries, with different incidences and death rates among regions/ethnicities and, intriguingly, between sexes. In addition to the many factors that can influence these discrepancies, we suggest a biological aspect, the genetic variation at the viral S protein receptor in human cells, ACE2 (angiotensin I-converting enzyme 2), which may contribute to the worse clinical outcome in males and in some regions worldwide. We performed exomics analysis in native and admixed South American populations, and we also conducted in silico genomics databank investigations in populations from other continents. Interestingly, at least ten polymorphisms in coding, noncoding and regulatory sites were found that can shed light on this issue and offer a plausible biological explanation for these epidemiological differences. In conclusion, there are ACE2 polymorphisms that could influence epidemiological discrepancies observed among ancestry and, moreover, between sexes.