ePyDGGA: automatic configuration for fitting epidemic curves.

Many epidemiological models and algorithms are used to fit the parameters of a given epidemic curve. On many occasions, fitting algorithms are interleaved with the actual epidemic models, which yields combinations of model-parameters that are hard to compare among themselves. Here, we provide a model-agnostic framework for epidemic parameter fitting that can (fairly) compare different epidemic models without jeopardizing the quality of the fitted parameters. Briefly, we have developed a Python framework that expects a Python function (epidemic model) and epidemic data and performs parameter fitting using automatic configuration. Our framework is capable of fitting parameters for any type of epidemic model, as long as it is provided as a Python function (or even in a different programming language). Moreover, we provide the code for different types of models, as well as the implementation of 4 concrete models with data to fit them. Documentation, code and examples can be found at https://ulog.udl.cat/static/doc/epidemic-gga/html/index.html .

Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation.

Chronic inflammation is a major cause of disease. Inflammation resolution is in part directed by the differential stability of mRNAs encoding pro-inflammatory and anti-inflammatory factors. In particular, tristetraprolin (TTP)-directed mRNA deadenylation destabilizes AU-rich element (ARE)-containing mRNAs. However, this mechanism alone cannot explain the variety of mRNA expression kinetics that are required to uncouple degradation of pro-inflammatory mRNAs from the sustained expression of anti-inflammatory mRNAs. Here, we show that the RNA-binding protein CPEB4 acts in an opposing manner to TTP in macrophages: it helps to stabilize anti-inflammatory transcripts harboring cytoplasmic polyadenylation elements (CPEs) and AREs in their 3'-UTRs, and it is required for the resolution of the lipopolysaccharide (LPS)-triggered inflammatory response. Coordination of CPEB4 and TTP activities is sequentially regulated through MAPK signaling. Accordingly, CPEB4 depletion in macrophages impairs inflammation resolution in an LPS-induced sepsis model. We propose that the counterbalancing actions of CPEB4 and TTP, as well as the distribution of CPEs and AREs in their target mRNAs, define transcript-specific decay patterns required for inflammation resolution. Thus, these two opposing mechanisms provide a fine-tuning control of inflammatory transcript destabilization while maintaining the expression of the negative feedback loops required for efficient inflammation resolution; disruption of this balance can lead to disease.

MoiRNAiFold: a novel tool for complex in silico RNA design.

Novel tools for in silico design of RNA constructs such as riboregulators are required in order to reduce time and cost to production for the development of diagnostic and therapeutic advances. Here, we present MoiRNAiFold, a versatile and user-friendly tool for de novo synthetic RNA design. MoiRNAiFold is based on Constraint Programming and it includes novel variable types, heuristics and restart strategies for Large Neighborhood Search. Moreover, this software can handle dozens of design constraints and quality measures and improves features for RNA regulation control of gene expression, such as Translation Efficiency calculation. We demonstrate that MoiRNAiFold outperforms any previous software in benchmarking structural RNA puzzles from EteRNA. Importantly, with regard to biologically relevant RNA designs, we focus on RNA riboregulators, demonstrating that the designed RNA sequences are functional both in vitro and in vivo. Overall, we have generated a powerful tool for de novo complex RNA design that we make freely available as a web server (https://moiraibiodesign.com/design/).

The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation.

Gemin5 is a predominantly cytoplasmic protein that downregulates translation, beyond controlling snRNPs assembly. The C-terminal region harbors a non-canonical RNA-binding site consisting of two domains, RBS1 and RBS2, which differ in RNA-binding capacity and the ability to modulate translation. Here, we show that these domains recognize distinct RNA targets in living cells. Interestingly, the most abundant and exclusive RNA target of the RBS1 domain was Gemin5 mRNA. Biochemical and functional characterization of this target demonstrated that RBS1 polypeptide physically interacts with a predicted thermodynamically stable stem-loop upregulating mRNA translation, thereby counteracting the negative effect of Gemin5 protein on global protein synthesis. In support of this result, destabilization of the stem-loop impairs the stimulatory effect on translation. Moreover, RBS1 stimulates translation of the endogenous Gemin5 mRNA. Hence, although the RBS1 domain downregulates global translation, it positively enhances translation of RNA targets carrying thermodynamically stable secondary structure motifs. This mechanism allows fine-tuning the availability of Gemin5 to play its multiple roles in gene expression control.

SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data.

RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our results support a newly identified partially double-stranded UUUUUGAGA motif similar to that known for the splicing factor HNRNPC.

The discovery potential of RNA processing profiles.

Small non-coding RNAs (sncRNAs) are highly abundant molecules that regulate essential cellular processes and are classified according to sequence and structure. Here we argue that read profiles from size-selected RNA sequencing capture the post-transcriptional processing specific to each RNA family, thereby providing functional information independently of sequence and structure. We developed SeRPeNT, a new computational method that exploits reproducibility across replicates and uses dynamic time-warping and density-based clustering algorithms to identify, characterize and compare sncRNAs by harnessing the power of read profiles. We applied SeRPeNT to: (i) generate an extended human annotation with 671 new sncRNAs from known classes and 131 from new potential classes, (ii) show pervasive differential processing of sncRNAs between cell compartments and (iii) predict new molecules with miRNA-like behaviour from snoRNA, tRNA and long non-coding RNA precursors, potentially dependent on the miRNA biogenesis pathway. Furthermore, we validated experimentally four predicted novel non-coding RNAs: a miRNA, a snoRNA-derived miRNA, a processed tRNA and a new uncharacterized sncRNA. SeRPeNT facilitates fast and accurate discovery and characterization of sncRNAs at an unprecedented scale. SeRPeNT code is available under the MIT license at https://github.com/comprna/SeRPeNT.

A novel translational control mechanism involving RNA structures within coding sequences.

The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.

RNAdualPF: software to compute the dual partition function with sample applications in molecular evolution theory.

BACKGROUND: RNA inverse folding is the problem of finding one or more sequences that fold into a user-specified target structure s 0, i.e. whose minimum free energy secondary structure is identical to the target s 0. Here we consider the ensemble of all RNA sequences that have low free energy with respect to a given target s 0. RESULTS: We introduce the program RNAdualPF, which computes the dual partition function Z ∗, defined as the sum of Boltzmann factors exp(-E(a,s 0)/RT) of all RNA nucleotide sequences a compatible with target structure s 0. Using RNAdualPF, we efficiently sample RNA sequences that approximately fold into s 0, where additionally the user can specify IUPAC sequence constraints at certain positions, and whether to include dangles (energy terms for stacked, single-stranded nucleotides). Moreover, since we also compute the dual partition function Z ∗(k) over all sequences having GC-content k, the user can require that all sampled sequences have a precise, specified GC-content. Using Z ∗, we compute the dual expected energy 〈E ∗〉, and use it to show that natural RNAs from the Rfam 12.0 database have higher minimum free energy than expected, thus suggesting that functional RNAs are under evolutionary pressure to be only marginally thermodynamically stable. We show that C. elegans precursor microRNA (pre-miRNA) is significantly non-robust with respect to mutations, by comparing the robustness of each wild type pre-miRNA sequence with 2000 [resp. 500] sequences of the same GC-content generated by RNAdualPF, which approximately [resp. exactly] fold into the wild type target structure. We confirm and strengthen earlier findings that precursor microRNAs and bacterial small noncoding RNAs display plasticity, a measure of structural diversity. CONCLUSION: We describe RNAdualPF, which rapidly computes the dual partition function Z ∗ and samples sequences having low energy with respect to a target structure, allowing sequence constraints and specified GC-content. Using different inverse folding software, another group had earlier shown that pre-miRNA is mutationally robust, even controlling for compositional bias. Our opposite conclusion suggests a cautionary note that computationally based insights into molecular evolution may heavily depend on the software used. C/C++-software for RNAdualPF is available at http://bioinformatics.bc.edu/clotelab/RNAdualPF .

RNAiFold2T: Constraint Programming design of thermo-IRES switches.

MOTIVATION: RNA thermometers (RNATs) are cis-regulatory elements that change secondary structure upon temperature shift. Often involved in the regulation of heat shock, cold shock and virulence genes, RNATs constitute an interesting potential resource in synthetic biology, where engineered RNATs could prove to be useful tools in biosensors and conditional gene regulation. RESULTS: Solving the 2-temperature inverse folding problem is critical for RNAT engineering. Here we introduce RNAiFold2T, the first Constraint Programming (CP) and Large Neighborhood Search (LNS) algorithms to solve this problem. Benchmarking tests of RNAiFold2T against existent programs (adaptive walk and genetic algorithm) inverse folding show that our software generates two orders of magnitude more solutions, thus allowing ample exploration of the space of solutions. Subsequently, solutions can be prioritized by computing various measures, including probability of target structure in the ensemble, melting temperature, etc. Using this strategy, we rationally designed two thermosensor internal ribosome entry site (thermo-IRES) elements, whose normalized cap-independent translation efficiency is approximately 50% greater at 42 °C than 30 °C, when tested in reticulocyte lysates. Translation efficiency is lower than that of the wild-type IRES element, which on the other hand is fully resistant to temperature shift-up. This appears to be the first purely computational design of functional RNA thermoswitches, and certainly the first purely computational design of functional thermo-IRES elements. AVAILABILITY: RNAiFold2T is publicly available as part of the new release RNAiFold3.0 at https://github.com/clotelab/RNAiFold and http://bioinformatics.bc.edu/clotelab/RNAiFold, which latter has a web server as well. The software is written in C ++ and uses OR-Tools CP search engine. CONTACT: clote@bc.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements.

The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation.

Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2(nmf205)(-/-) mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNA(Arg)UCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2(nmf205)(-/-) mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress.

Genetic Architectures of Quantitative Variation in RNA Editing Pathways.

RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.

RNAiFold 2.0: a web server and software to design custom and Rfam-based RNA molecules.

UNLABELLED: Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. AVAILABILITY: the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0.

Segmentation and tracking of adherens junctions in 3D for the analysis of epithelial tissue morphogenesis.

Epithelial morphogenesis generates the shape of tissues, organs and embryos and is fundamental for their proper function. It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics, to cell deformations, mitosis and apoptosis, to coordinated cell rearrangements that lead to global changes of tissue shape. Using time lapse imaging, it is possible to observe these events at a system level. However, to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data. Toward this goal, we developed an image-based computational pipeline to preprocess, segment and track epithelial cells in 4D confocal microscopy data. The computational pipeline we developed, for the first time, detects the adherens junctions of epithelial cells in 3D, without the need to first detect cell nuclei. We accentuate and detect cell outlines in a series of steps, symbolically describe the cells and their connectivity, and employ this information to track the cells. We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts, track cells, and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs. We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis. We have made our methods and data available as an open-source multiplatform software tool called TTT (http://github.com/morganrcu/TTT).

RNA folding pathways and kinetics using 2D energy landscapes.

RNA folding pathways play an important role in various biological processes, such as (i) the hok/sok (host-killing/suppression of killing) system in E. coli to check for sufficient plasmid copy number, (ii) the conformational switch in spliced leader (SL) RNA from Leptomonas collosoma, which controls trans splicing of a portion of the '5 exon, and (iii) riboswitches--portions of the 5' untranslated region of messenger RNA that regulate genes by allostery. Since RNA folding pathways are determined by the energy landscape, we describe a novel algorithm, FFTbor2D, which computes the 2D projection of the energy landscape for a given RNA sequence. Given two metastable secondary structures A, B for a given RNA sequence, FFTbor2D computes the Boltzmann probability p(x, y) = Z(x,y)/Z that a secondary structure has base pair distance x from A and distance y from B. Using polynomial interpolationwith the fast Fourier transform,we compute p(x, y) in O(n(5)) time and O(n(2)) space, which is an improvement over an earlier method, which runs in O(n(7)) time and O(n(4)) space. FFTbor2D has potential applications in synthetic biology, where one might wish to design bistable switches having target metastable structures A, B with favorable pathway kinetics. By inverting the transition probability matrix determined from FFTbor2D output, we show that L. collosoma spliced leader RNA has larger mean first passage time from A to B on the 2D energy landscape, than 97.145% of 20,000 sequences, each having metastable structures A, B. Source code and binaries are freely available for download at http://bioinformatics.bc.edu/clotelab/FFTbor2D. The program FFTbor2D is implemented in C++, with optional OpenMP parallelization primitives.

Complete RNA inverse folding: computational design of functional hammerhead ribozymes.

Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/.

RNA function. Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.

In higher eukaryotes, transfer RNAs (tRNAs) with the same anticodon are encoded by multiple nuclear genes, and little is known about how mutations in these genes affect translation and cellular homeostasis. Similarly, the surveillance systems that respond to such defects in higher eukaryotes are not clear. Here, we discover that loss of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, in mice with a mutation in a tRNA gene that is specifically expressed in the central nervous system causes ribosome stalling and widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor but also unmask the disease potential of mutations in nuclear-encoded tRNA genes.

Energy parameters and novel algorithms for an extended nearest neighbor energy model of RNA.

We describe the first algorithm and software, RNAenn, to compute the partition function and minimum free energy secondary structure for RNA with respect to an extended nearest neighbor energy model. Our next-nearest-neighbor triplet energy model appears to lead to somewhat more cooperative folding than does the nearest neighbor energy model, as judged by melting curves computed with RNAenn and with two popular software implementations for the nearest-neighbor energy model. A web server is available at http://bioinformatics.bc.edu/clotelab/RNAenn/.