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The vast majority of viruses consist of a nucleic acid surrounded by a protective icosahedral protein shell called the capsid. During viral infection of a host cell, the timing and efficiency of the assembly process is important for ensuring the production of infectious new progeny virus particles. In the class of single-stranded RNA (ssRNA) viruses, the assembly of the capsid takes place in tandem with packaging of the ssRNA genome in a highly cooperative co-assembly process. In simple ssRNA viruses such as the bacteriophage MS2 and small RNA plant viruses such as STNV, this cooperative process results from multiple interactions between the protein shell and sites in the RNA genome which have been termed packaging signals. Using a stochastic assembly algorithm which includes cooperative interactions between the protein shell and packaging signals in the RNA genome, we demonstrate that highly efficient assembly of STNV capsids arises from a set of simple local rules. Altering the local assembly rules results in different nucleation scenarios with varying assembly efficiencies, which in some cases depend strongly on interactions with RNA packaging signals. Our results provide a potential simple explanation based on local assembly rules for the ability of some ssRNA viruses to spontaneously assemble around charged polymers and other non-viral RNAs in vitro., Author Summary Assembly in single-stranded RNA plant viruses takes place via a highly cooperative process in which capsid proteins co-assemble around ssRNA. In the small satellite plant virus STNV, small hairpins present in the genome, termed packaging signals, bind to capsid proteins during assembly and allow for efficient formation of the capsid shell. Although these hairpins have been visualised in X-ray crystallography, the local rules of their interaction with the capsid proteins and how they ensure an efficient assembly process is somewhat unknown. Here we test several assembly scenarios involving different local rules for the protein-protein and RNA-protein interactions and find that assembly efficiency is highly dependent on the local assembly rules. Interestingly, while certain local assembly rules are consistent with a packaging signal mediated assembly model, some local rules predict reasonable assembly efficiency independent of packaging signal distribution. This may explain the ability to package charged polymer materials in some plant viruses.

Deep neural networks have demonstrated improved performance at predicting the sequence specificities of DNA- and RNA-binding proteins compared to previous methods that rely on k-mers and position weight matrices. To gain insights into why a DNN makes a given prediction, model interpretability methods, such as attribution methods, can be employed to identify motif-like representations along a given sequence. Because explanations are given on an individual sequence basis and can vary substantially across sequences, deducing generalizable trends across the dataset and quantifying their effect size remains a challenge. Here we introduce global importance analysis (GIA), a model interpretability method that quantifies the population-level effect size that putative patterns have on model predictions. GIA provides an avenue to quantitatively test hypotheses of putative patterns and their interactions with other patterns, as well as map out specific functions the network has learned. As a case study, we demonstrate the utility of GIA on the computational task of predicting RNA-protein interactions from sequence. We first introduce a convolutional network, we call ResidualBind, and benchmark its performance against previous methods on RNAcompete data. Using GIA, we then demonstrate that in addition to sequence motifs, ResidualBind learns a model that considers the number of motifs, their spacing, and sequence context, such as RNA secondary structure and GC-bias., Author summary Although deep neural networks are becoming widely applied in genomics, it remains unclear why they make a given prediction. For model interpretability, attribution methods reveal the independent importance of single nucleotide variants in a given sequence on model predictions. While the resultant attribution maps can help to identify representations of motifs, it remains challenging to identify generalizable patterns across the dataset and to quantify their effect size on model predictions. Here, we introduce an interpretability method called global importance analysis (GIA) to quantify the effect size that putative patterns have on model predictions across a population of sequences. GIA provides a natural follow up to current interpretability methods to quantitatively test hypotheses of putative patterns (and their interactions with other patterns). As a case study, we demonstrate how it can be used for the computational task of predicting RNA-protein interactions and show that deep learning models can learn not only sequence motifs, but also the number of motifs, their spacing, and sequence context, such as RNA secondary structure and GC-bias.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5’-viral genome-linked protein (VPg) and have a 3’ polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3’ untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5’ regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5’ region and 3’ UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5’ region and 3’ UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5’ proximal coding region of the RNA2 polyprotein and in the RNA2 3’ UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3’ UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3’ UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5’ region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773–972) of the 1547 nt long 3’ UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2’s own RNA evades nsp1’s host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results suggested that the stem loop structure of the SARS-CoV-2 RNA 5’-untranslated region (SL1) binds to both nsp1’s N-terminal globular region and intrinsically disordered region. The consistency of the results was assessed by modeling nsp1-40S ribosome structure based on reported nsp1 experiments, including the X-ray crystallographic structure analysis, the cryo-EM electron density map, and cross-linking experiments. The SL1 binding region predicted from the simulation was open to the solvent, yet the ribosome could interact with SL1. Cluster analysis of the binding mode and detailed analysis of the binding poses suggest residues Arg124, Lys47, Arg43, and Asn126 may be involved in the SL1 recognition mechanism, consistent with the existing mutational analysis., Author summary The pandemic of COVID-19 is still rampant all over the world as of 2021 June. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative pathogen of COVID-19, encodes a protein called nsp1 (nonstructural protein 1), which modulates and hijacks the ribosome of the infected host cells. With nsp1, infected human cells selectively translate SARS-CoV-2’s RNA, which increases the virus reproduction efficiency while evading the host immunity. Though it has been known that nsp1 recognizes characteristic stem-loop structure at 5’-end of SARS-CoV-2’s RNA (called SL1), the molecular mechanism underlying the recognition has been poorly understood. We investigated the mechanism of selective translation using the all-atom molecular dynamics simulation of nsp1-SL1 complex. Our simulation results suggest that the binding between nsp1 and SL1 is multi-modal. The results also imply that both the N-terminal globular part and the C-terminal flexible tail of nsp1 are involved in the binding. The residues involved in nsp1-SL1 binding coincides with the known mutant analyses of SARS-CoV-1 and SARS-CoV-2, as well as experimental evidence about nsp1-ribosome interactions.

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure., Author summary Potato spindle tuber viroid (PSTVd) is a small, circular non-coding RNA that systemically infects host plants. All functions required for replication as well as cell-to-cell and systemic spread are mediated by the RNA genome, whose 2D structure consists of multiple base paired stems and loops. However, a wealth of evidence indicates that most loops in RNA molecules are structured by non-canonical base pairs and other interactions. Here, we show that PSTVd loop 27 forms a 3D structure that is essential for replication. Analysis of an exceptional mutant revealed that loop 27 is also required for transit from infected epidermal cells to underlying palisade mesophyll cells, but is not needed for transport in the reverse direction or into other cell types. Thus, loop 27 is necessary for epidermal exit but not entry, and requirements for transit between epidermal cells and palisade mesophyll cells are unique and directional. PSTVd moves between cells through specialized structures known as plasmodesmata. Combined with previous findings, these studies suggest that plasmodesmal gates interconnecting most and perhaps all plant cell types are unique, allowing precise regulation of RNA transport and the establishment of distinct cellular boundaries.

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world., Author summary RNAs frequently misfold into stable but biologically inactive structures especially under stresses, while RNA chaperones interact with various RNAs to prevent the structures that may cause premature transcriptional termination or pausing, and affect mRNA decay and translation. This work for the first time reports that an archaeal RNA chaperone TRAM0076 globally affects the transcription of methanogenic Archaea through posttranscriptional actions. TRAM0076 also binds many cellular mRNAs, possibly at the 5′-untranslated regions. This work uncovers an important regulatory element of ancient life in the RNA world.

Acetamiprid (ACE) is a kind of broad-spectrum pesticide that has potential health risk to human beings. Aptamers (Ap-DNA (1)) have a great potential as analytical tools for pesticide detection. In this work, a label-free electrochemical sensing assay for ACE determination is presented by electrochemical impedance spectroscopy (EIS). And the specific binding model between ACE and Ap-DNA (1) was further investigated for the first time. Circular dichroism (CD) spectroscopy and EIS demonstrated that the single strand AP-DNA (1) first formed a loosely secondary structure in Tris-HClO4 (20 mM, pH = 7.4), and then transformed into a more stable hairpin-like structure when incubated in binding buffer (B-buffer). The formed stem-loop bulge provides the specific capturing sites for ACE, forming ACE/AP-DNA (1) complex, and induced the RCT (charge transfer resistance) increase between the solution-based redox probe [Fe(CN)6]3−/4− and the electrode surface. The change of ΔRCT (charge transfer resistance change, ΔRCT = RCT(after)-RCT(before)) is positively related to the ACE level. As a result, the AP-DNA (1) biosensor showed a high sensitivity with the ACE concentration range spanning from 5 nM to 200 mM and a detection limit of 1 nM. The impedimetric AP-DNA (1) sensor also showed good selectivity to ACE over other selected pesticides and exhbited excellent performance in environmental water and orange juice samples analysis, with spiked recoveries in the range of 85.8% to 93.4% in lake water and 83.7% to 89.4% in orange juice. With good performance characteristics of practicality, sensitivity and selectivity, the AP-DNA (1) sensor holds a promising application for the on-site ACE detection.

Motivation RNA is a biopolymer with various applications inside the cell and in biotechnology. Structure of an RNA molecule mainly determines its function and is essential to guide nanostructure design. Since experimental structure determination is time-consuming and expensive, accurate computational prediction of RNA structure is of great importance. Prediction of RNA secondary structure is relatively simpler than its tertiary structure and provides information about its tertiary structure, therefore, RNA secondary structure prediction has received attention in the past decades. Numerous methods with different folding approaches have been developed for RNA secondary structure prediction. While methods for prediction of RNA pseudoknot-free structure (structures with no crossing base pairs) have greatly improved in terms of their accuracy, methods for prediction of RNA pseudoknotted secondary structure (structures with crossing base pairs) still have room for improvement. A long-standing question for improving the prediction accuracy of RNA pseudoknotted secondary structure is whether to focus on the prediction algorithm or the underlying energy model, as there is a trade-off on computational cost of the prediction algorithm versus the generality of the method. Results The aim of this work is to argue when comparing different methods for RNA pseudoknotted structure prediction, the combination of algorithm and energy model should be considered and a method should not be considered superior or inferior to others if they do not use the same scoring model. We demonstrate that while the folding approach is important in structure prediction, it is not the only important factor in prediction accuracy of a given method as the underlying energy model is also as of great value. Therefore we encourage researchers to pay particular attention in comparing methods with different energy models.

Restriction enzymes are the bread and butter of Molecular Biology. Nonetheless, how restriction enzymes recognize and cleave their target is not always clear. When developing a method for the enzymatic production of oligonucleotides, we noticed that type II endonucleases BtsCI and BseGI, which recognize the sequence GGATGNN^, perform incomplete digestions of DNA hairpins, with the top strand nick not always occurring correctly. We tested the cutting of synthetic hairpins containing all possible combinations of dinucleotides following the recognition site and our results show that all sequences containing one adenine following GGATG were digested more efficiently. We further show that the same sequence preference is also observable in double stranded DNA at higher Mg2+ concentrations and even in optimal conditions. Kinetic results show that BtsCI has a noteworthy difference in the first-rate constants between different sequences and between the two catalytic domains. An increase in Mg2+ resulted in a drastic decrease in the catalytic activity of the top (sense) strand that wasn’t always accompanied by a nick in the bottom strand (antisense).

Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α–helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein “halves” for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.

The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss-of-function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that microRNA (miRNA)-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a consensus gene signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 single guide RNAs (sgRNAs) in 6 cells lines and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function., Author summary The Connectivity Map (CMAP) is a large-scale resource of the gene expression changes caused by small-molecule and genetic perturbations in multiple cell types. Here, we analyze 2 types of genetic perturbations, RNAi and CRISPR, using CMAP data. RNAi and CRISPR are both used to generate loss-of-function mutants but exploit different cellular pathways. We find that the on-target efficacy of the 2 technologies is similar. However, the off-target effects of RNAi are much greater than typically appreciated, whereas CRISPR technology has negligible off-target activity. To enhance the use of CMAP data generated with genetic reagents, we develop the consensus gene signature (CGS) and demonstrate that it can improve the identification of on-target activity, which will be particularly critical for analyzing data generated with RNAi. Overall, the large-scale data generated as part of CMAP allow us to robustly characterize the on- and off-target properties of 2 common approaches for genetic manipulation of cells.

We have reported previously that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3'UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage.

During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonu- clease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs, including the transcript encoding interleukin-6 (IL-6), escape SOX-induced cleavage. IL-6 escape is mediated through a 3’ UTR RNA regulatory element that overrides the SOX targeting mechanism. Here, we reveal that this protective RNA element functions to broadly restrict cleavage by a range of homologous and non-homologous viral endonucleases. However, it does not impede cleavage by cellular endonucleases. The IL-6 protective sequence may be representative of a larger class of nuclease escape elements, as we identified a similar protective element in the GADD45B mRNA. The IL-6 and GADD45B-derived elements display similarities in their sequence, putative structure, and several associated RNA binding proteins. However, the overall composition of their ribonucleoprotein complexes appears distinct, leading to differences in the breadth of nucleases restricted. These findings highlight how RNA elements can selectively control transcript abundance in the background of widespread virus-induced mRNA degradation., Author summary The ability of viruses to control the host gene expression environment is crucial to promote viral infection. Many viruses express factors that reduce host gene expression through widespread mRNA decay. However, some mRNAs escape this fate, like the transcript encoding the immunoregulatory cytokine IL-6 during KSHV infection. IL-6 escape relies on an RNA regulatory element located in its 3’UTR and involves the recruitment of a protective protein complex. Here, we show that this escape extends beyond KSHV to a variety of related and unrelated viral endonucleases. However, the IL-6 element does not protect against cellular endonucleases, revealing for the first time a virus-specific nuclease escape element. We identified a related escape element in the GADD45B mRNA, which displays several similarities with the IL-6 element. However, these elements assemble a largely distinct complex of proteins, leading to differences in the breadth of their protective capacity. Collectively, these findings reveal how a putative new class of RNA elements function to control RNA fate in the background of widespread mRNA degradation by viral endonucleases.

We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5' untranslated region (UTR) of Triticum mosaic virus (TriMV, Potyviridae). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5' UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5' UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome., Author summary While the computational prediction of microRNA loci from high-throughput sequence data is well-studied, challenges persist in defining the minimum number of reads required for a locus to be evaluated, as well as in defining the precursor span. We present a new method, “miRWoods”, which has greater recall of known microRNAs, while also achieving as good or better overall performance. Our approach uses improved duplex-based methods of precursor detection and a pair of random forest layers that sensitively detect mature products and precursors. We trained our model on data from human, and confirmed that it can successfully be applied cross-species by evaluating predictions for the mouse genome. We then applied our approach to new sequencing data mapped to the under-annotated genomes of cow and cat. We were able to use miRWoods to improve annotations for cat and cow microRNAs, and found novel microRNAs in human and mouse, and identified errors in current annotations.

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression., This study reveals a novel role for the calcium-binding protein calreticulin in the survival of cancer cells; downregulation of calreticulin leads to mitochondrial calcium overload and an induction of non-apoptotic cell death. Calreticulin levels inversely correlate with the survival of patients diagnosed with various types of solid cancers.

Tauopathies are neurodegenerative diseases that affect millions of people worldwide including those with Alzheimer’s disease. While many efforts have focused on understanding the role of tau protein in neurodegeneration, there has been little done to systematically analyze and study the structures within tau’s encoding RNA and their connection to disease pathology. Knowledge of RNA structure can provide insights into disease mechanisms and how to affect protein production for therapeutic benefit. Using computational methods based on thermodynamic stability and evolutionary conservation, we identified structures throughout the tau pre-mRNA, especially at exon-intron junctions and within the 5′ and 3′ untranslated regions (UTRs). In particular, structures were identified at twenty exon-intron junctions. The 5′ UTR contains one structured region, which lies within a known internal ribosome entry site. The 3′ UTR contains eight structured regions, including one that contains a polyadenylation signal. A series of functional experiments were carried out to assess the effects of mutations associated with mis-regulation of alternative splicing of exon 10 and to identify regions of the 3′ UTR that contain cis-regulatory elements. These studies defined novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases.

Mounting evidence shows mammalian brains are probabilistic computers, but the specific cells involved remain elusive. Parallel research suggests that grid cells of the mammalian hippocampal formation are fundamental to spatial cognition but their diverse response properties still defy explanation. No plausible model exists which explains stable grids in darkness for twenty minutes or longer, despite being one of the first results ever published on grid cells. Similarly, no current explanation can tie together grid fragmentation and grid rescaling, which show very different forms of flexibility in grid responses when the environment is varied. Other properties such as attractor dynamics and grid anisotropy seem to be at odds with one another unless additional properties are assumed such as a varying velocity gain. Modelling efforts have largely ignored the breadth of response patterns, while also failing to account for the disastrous effects of sensory noise during spatial learning and recall, especially in darkness. Here, published electrophysiological evidence from a range of experiments are reinterpreted using a novel probabilistic learning model, which shows that grid cell responses are accurately predicted by a probabilistic learning process. Diverse response properties of probabilistic grid cells are statistically indistinguishable from rat grid cells across key manipulations. A simple coherent set of probabilistic computations explains stable grid fields in darkness, partial grid rescaling in resized arenas, low-dimensional attractor grid cell dynamics, and grid fragmentation in hairpin mazes. The same computations also reconcile oscillatory dynamics at the single cell level with attractor dynamics at the cell ensemble level. Additionally, a clear functional role for boundary cells is proposed for spatial learning. These findings provide a parsimonious and unified explanation of grid cell function, and implicate grid cells as an accessible neuronal population readout of a set of probabilistic spatial computations., Author Summary Cells in the mammalian hippocampal formation are thought to be central for spatial learning and stable spatial representations. Of the known spatial cells, grid cells form strikingly regular and stable patterns of activity, even in darkness. Hence, grid cells may provide the universal metric upon which spatial cognition is based. However, a more fundamental problem is how grids themselves may form and stabilise, since sensory information is noisy and can vary tremendously with environmental conditions. Furthermore, the same grid cell can display substantially different yet stable patterns of activity in different environments. Currently, no model explains how vastly different sensory cues can give rise to the diverse but stable grid patterns. Here, a new probabilistic model is proposed which combines information encoded by grid cells and boundary cells. This noise-tolerant model performs robust spatial learning, under a variety of conditions, and produces varied yet stable grid cell response patterns like rodent grid cells. Across numerous experimental manipulations, rodent and probabilistic grid cell responses are similar or even statistically indistinguishable. These results complement a growing body of evidence suggesting that mammalian brains are inherently probabilistic, and suggest for the first time that grid cells may be involved.

Micro RNAs (miRNAs) (19-25 nucleotides in length) belong to the group of non-coding RNAs are the most abundant group of posttranscriptional regulators in multicellular organisms. They affect a gene expression by binding of fully or partially complementary sequences to the 3'-UTR of target mRNA. Furthermore, miRNAs present a mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated at the post-transcriptional level. However, little is known about the specific pathways through which miRNAs with specific sequence or structural motifs regulate the cellular processes. In this paper we showed the broad and deep characteristics of mature miRNAs according to their sequence and structural motifs. We investigated a distinct group of miRNAs characterized by the presence of specific sequence motifs, such as UGUGU, GU-repeats and purine/pyrimidine contents. Using computational function and pathway analysis of their targeted genes, we were able to observe the relevance of sequence and the type of targeted mRNAs. As the consequence of the sequence analysis we finally provide the comprehensive description of pathways, biological processes and proteins associated with the distinct group of characterized miRNAs. Here, we found that the specific group of miRNAs with UGUGU can activate the targets associated to the interferon induction pathway or pathways prominently observed during carcinogenesis. GU-rich miRNAs are prone to regulate mostly processes in neurogenesis, whereas purine/pyrimidine rich miRNAs could be involved rather in transport and/or degradation of RNAs. Additionally, we have also analyzed the simple sequence repeats (SSRs). Their variation within mature miRNAs might be critical for normal miRNA regular activity. Expansion or contraction of SSRs in mature miRNA might directly affect its mRNA interaction or even change the function of that distinct miRNA. Our results prove that due to the specific sequence features, these molecules can also be involved in well-defined cellular processes depending on their sequence contents. The pathway mapping and theoretical gene target identification allowed us to create a biological framework to show the relevance of the specific miRNAs in regulation the distinct type of targets.

The aggregation of amyloid-β peptides is associated with the pathogenesis of Alzheimer's disease, in which the 30-36 fragments play an important part as a fiber-forming hydrophobic region. The fibrillar structure of Aβ30-36 has been detected by means of X-ray diffraction, but its oligomeric structural determination, biophysical characterization, and pathological mechanism remain elusive. In this study, we have investigated the structures of Aβ30-36 hexamer as well as its G33V and L34T mutants in explicit water environment using replica-exchange molecular dynamics (REMD) simulations. Our results show that the wild-type (WT) Aβ30-36 hexamer has a preference to form β-barrel and bilayer β-sheet conformations, while the G33V or L34T mutation disrupts the β-barrel structures: the G33V mutant is homogenized to adopt β-sheet-rich bilayers, and the structures of L34T mutant on the contrary get more diverse. The hydrophobic interaction plays a critical role in the formation and stability of oligomeric assemblies among all the three systems. In addition, the substitution of G33 by V reduces the β-sheet content in the most populated conformations of Aβ30-36 oligomers through a steric effect. The L34T mutation disturbs the interpeptide hydrogen bonding network, and results in the increased coil content and morphological diversity. Our REMD runs provide structural details of WT and G33V/L34T mutant Aβ30-36 oligomers, and molecular insight into the aggregation mechanism, which will be helpful for designing novel inhibitors or amyloid-based materials.

MicroRNA play an important role in post-transcriptional regulation of most transcripts in the human genome, but their evolution across the primate lineage is largely uncharacterized. A particular miRNA can have one to thousands of messenger RNA targets, establishing the potential for a small change in sequence or overall miRNA structure to have profound phenotypic effects. However, the majority of non-human primate miRNA is predicted solely by homology to the human genome and lacks experimental validation. In the present study, we sequenced thirteen species representing a wide range of the primate phylogeny. Hundreds of miRNA were validated, and the number of species with experimentally validated miRNA was tripled. These species include a sister taxon to humans (bonobo) and basal primates (aye-aye, mouse lemur, galago). Consistent with previous studies, we found the seed region and mature miRNA to be highly conserved across primates, with overall structural conservation of the pre-miRNA hairpin. However, there were a number of interesting exceptions, including a seed shift due to structural changes in miR-501. We also identified an increase in the number of miR-320 paralogs throughout primate evolution. Many of these non-conserved miRNA appear to regulate neuronal processes, illustrating the importance of investigating miRNA to learn more about human evolution.

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.