Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.

Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Alteration of microglial metabolism and inflammatory profile contributes to neurotoxicity in a hiPSC-derived microglia model of frontotemporal dementia 3.

Frontotemporal dementia (FTD) is a common cause of early-onset dementia, with no current treatment options. FTD linked to chromosome 3 (FTD3) is a rare sub-form of the disease, caused by a point mutation in the Charged Multivesicular Body Protein 2B (CHMP2B). This mutation causes neuronal phenotypes, such as mitochondrial deficiencies, accompanied by metabolic changes and interrupted endosomal-lysosomal fusion. However, the contribution of glial cells to FTD3 pathogenesis has, until recently, been largely unexplored. Glial cells play an important role in most neurodegenerative disorders as drivers and facilitators of neuroinflammation. Microglia are at the center of current investigations as potential pro-inflammatory drivers. While gliosis has been observed in FTD3 patient brains, it has not yet been systematically analyzed. In the light of this, we investigated the role of microglia in FTD3 by implementing human induced pluripotent stem cells (hiPSC) with either a heterozygous or homozygous CHMP2B mutation, introduced into a healthy control hiPSC line via CRISPR-Cas9 precision gene editing. These hiPSC were differentiated into microglia to evaluate the pro-inflammatory profile and metabolic state. Moreover, hiPSC-derived neurons were cultured with conditioned microglia media to investigate disease specific interactions between the two cell populations. Interestingly, we identified two divergent inflammatory microglial phenotypes resulting from the underlying mutations: a severe pro-inflammatory profile in CHMP2B homozygous FTD3 microglia, and an "unresponsive" CHMP2B heterozygous FTD3 microglial state. These findings correlate with our observations of increased phagocytic activity in CHMP2B homozygous, and impaired protein degradation in CHMP2B heterozygous FTD3 microglia. Metabolic mapping confirmed these differences, revealing a metabolic reprogramming of the CHMP2B FTD3 microglia, displayed as a compensatory up-regulation of glutamine metabolism in the CHMP2B homozygous FTD3 microglia. Intriguingly, conditioned CHMP2B homozygous FTD3 microglia media caused neurotoxic effects, which was not evident for the heterozygous microglia. Strikingly, IFN-γ treatment initiated an immune boost of the CHMP2B heterozygous FTD3 microglia, and conditioned microglia media exposure promoted neural outgrowth. Our findings indicate that the microglial profile, activity, and behavior is highly dependent on the status of the CHMP2B mutation. Our results suggest that the heterozygous state of the mutation in FTD3 patients could potentially be exploited in form of immune-boosting intervention strategies to counteract neurodegeneration.

RNA-Seq Study on the Longissimus thoracis Muscle of Italian Large White Pigs Fed Extruded Linseed with or without Antioxidants and Polyphenols.

The addition of n-3 polyunsaturated fatty acids (n-3 PUFAs) to the swine diet increases their content in muscle cells, and the additional supplementation of antioxidants promotes their oxidative stability. However, to date, the functionality of these components within muscle tissue is not well understood. Using a published RNA-seq dataset and a selective workflow, the study aimed to find the differences in gene expression and investigate how differentially expressed genes (DEGs) were implicated in the cellular composition and metabolism of muscle tissue of 48 Italian Large White pigs under different dietary conditions. A functional enrichment analysis of DEGs, using Cytoscape, revealed that the diet enriched with extruded linseed and supplemented with vitamin E and selenium promoted a more rapid and massive immune system response because the overall function of muscle tissue was improved, while those enriched with extruded linseed and supplemented with grape skin and oregano extracts promoted the presence and oxidative stability of n-3 PUFAs, increasing the anti-inflammatory potential of the muscular tissue.

The transcriptomic landscape of neurons carrying PSEN1 mutations reveals changes in extracellular matrix components and non-coding gene expression.

Alzheimer's disease (AD) is a progressive and irreversible brain disorder, which can occur either sporadically, due to a complex combination of environmental, genetic, and epigenetic factors, or because of rare genetic variants in specific genes (familial AD, or fAD). A key hallmark of AD is the accumulation of amyloid beta (Aß) and Tau hyperphosphorylated tangles in the brain, but the underlying pathomechanisms and interdependencies remain poorly understood. Here, we identify and characterise gene expression changes related to two fAD mutations (A79V and L150P) in the Presenilin-1 (PSEN1) gene. We do this by comparing the transcriptomes of glutamatergic forebrain neurons derived from fAD-mutant human induced pluripotent stem cells (hiPSCs) and their individual isogenic controls generated via precision CRISPR/Cas9 genome editing. Our analysis of Poly(A) RNA-seq data detects 1111 differentially expressed coding and non-coding genes significantly altered in fAD. Functional characterisation and pathway analysis of these genes reveal profound expression changes in constituents of the extracellular matrix, important to maintain the morphology, structural integrity, and plasticity of neurons, and in genes involved in calcium homeostasis and mitochondrial oxidative stress. Furthermore, by analysing total RNA-seq data we reveal that 30 out of 31 differentially expressed circular RNA genes are significantly upregulated in the fAD lines, and that these may contribute to the observed protein-coding gene expression changes. The results presented in this study contribute to a better understanding of the cellular mechanisms impacted in AD neurons, ultimately leading to neuronal damage and death.

Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

Stellate cells are principal neurons in the entorhinal cortex that contribute to spatial processing. They also play a role in the context of Alzheimer's disease as they accumulate Amyloid beta early in the disease. Producing human stellate cells from pluripotent stem cells would allow researchers to study early mechanisms of Alzheimer's disease, however, no protocols currently exist for producing such cells. In order to develop novel stem cell protocols, we characterize at high resolution the development of the porcine medial entorhinal cortex by tracing neuronal and glial subtypes from mid-gestation to the adult brain to identify the transcriptomic profile of progenitor and adult stellate cells. Importantly, we could confirm the robustness of our data by extracting developmental factors from the identified intermediate stellate cell cluster and implemented these factors to generate putative intermediate stellate cells from human induced pluripotent stem cells. Six transcription factors identified from the stellate cell cluster including RUNX1T1, SOX5, FOXP1, MEF2C, TCF4, EYA2 were overexpressed using a forward programming approach to produce neurons expressing a unique combination of RELN, SATB2, LEF1 and BCL11B observed in stellate cells. Further analyses of the individual transcription factors led to the discovery that FOXP1 is critical in the reprogramming process and omission of RUNX1T1 and EYA2 enhances neuron conversion. Our findings contribute not only to the profiling of cell types within the developing and adult brain's medial entorhinal cortex but also provides proof-of-concept for using scRNAseq data to produce entorhinal intermediate stellate cells from human pluripotent stem cells in-vitro.

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production.

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes via gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

The Bacillaceae-1 RNA motif comprises two distinct classes.

Non-coding RNAs are key regulatory players in bacteria. Many computationally predicted non-coding RNAs, however, lack functional associations. An example is the Bacillaceae-1 RNA motif, whose Rfam model consists of two hairpin loops. We find the motif conserved in nine of 13 non-pathogenic strains of the genus Bacillus but only in one pathogenic strain. To elucidate functional characteristics, we studied 118 hits of the Rfam model in 11 Bacillus spp. and found two distinct classes based on the ensemble diversity of their RNA secondary structure and the genomic context concerning the ribosomal RNA (rRNA) cluster. Forty hits are associated with the rRNA cluster, of which all 19 hits upstream flanking of 16S rRNA have a reverse complementary structure of low structural diversity. Fifty-two hits have large ensemble diversity, of which 38 are located between two coding genes. For eight hits in Bacillus subtilis, we investigated public expression data under various conditions and observed either the forward or the reverse complementary motif expressed. Five hits are associated with the rRNA cluster. Four of them are located upstream of the 16S rRNA and are not transcriptionally active, but instead, their reverse complements with low structural diversity are expressed together with the rRNA cluster. The three other hits are located between two coding genes in non-conserved genomic loci. Two of them are independently expressed from their surrounding genes and are structurally diverse. In summary, we found that Bacillaceae-1 RNA motifs upstream flanking of ribosomal RNA clusters tend to have one stable structure with the reverse complementary motif expressed in B. subtilis. In contrast, a subgroup of intergenic motifs has the thermodynamic potential for structural switches.

Does rapid sequence divergence preclude RNA structure conservation in vertebrates?

Accelerated evolution of any portion of the genome is of significant interest, potentially signaling positive selection of phenotypic traits and adaptation. Accelerated evolution remains understudied for structured RNAs, despite the fact that an RNA's structure is often key to its function. RNA structures are typically characterized by compensatory (structure-preserving) basepair changes that are unexpected given the underlying sequence variation, i.e., they have evolved through negative selection on structure. We address the question of how fast the primary sequence of an RNA can change through evolution while conserving its structure. Specifically, we consider predicted and known structures in vertebrate genomes. After careful control of false discovery rates, we obtain 13 de novo structures (and three known Rfam structures) that we predict to have rapidly evolving sequences-defined as structures where the primary sequences of human and mouse have diverged at least twice as fast (1.5 times for Rfam) as nearby neutrally evolving sequences. Two of the three known structures function in translation inhibition related to infection and immune response. We conclude that rapid sequence divergence does not preclude RNA structure conservation in vertebrates, although these events are relatively rare.

CRISPRroots: on- and off-target assessment of RNA-seq data in CRISPR-Cas9 edited cells.

The CRISPR-Cas9 genome editing tool is used to study genomic variants and gene knockouts, and can be combined with transcriptomic analyses to measure the effects of such alterations on gene expression. But how can one be sure that differential gene expression is due to a successful intended edit and not to an off-target event, without performing an often resource-demanding genome-wide sequencing of the edited cell or strain? To address this question we developed CRISPRroots: CRISPR-Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites. Our method combines Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells to discover potential off-targets. Applied on seven public datasets, CRISPRroots identified critical off-target candidates that were overlooked in all of the corresponding previous studies. CRISPRroots is available via https://rth.dk/resources/crispr.

Modelling Nonalcoholic Steatohepatitis In Vivo-A Close Transcriptomic Similarity Supports the Guinea Pig Disease Model.

The successful development of effective treatments against nonalcoholic steatohepatitis (NASH) is significantly set back by the limited availability of predictive preclinical models, thereby delaying and reducing patient recovery. Uniquely, the guinea pig NASH model develops hepatic histopathology and fibrosis resembling that of human patients, supported by similarities in selected cellular pathways. The high-throughput sequencing of guinea pig livers with fibrotic NASH (n = 6) and matched controls (n = 6) showed a clear separation of the transcriptomic profile between NASH and control animals. A comparison to NASH patients with mild disease (GSE126848) revealed a 45.2% overlap in differentially expressed genes, while pathway analysis showed a 34% match between the top 50 enriched pathways in patients with advanced NASH (GSE49541) and guinea pigs. Gene set enrichment analysis highlighted the similarity to human patients (GSE49541), also when compared to three murine models (GSE52748, GSE38141, GSE67680), and leading edge genes THRSP, CCL20 and CD44 were highly expressed in both guinea pigs and NASH patients. Nine candidate genes were identified as highly correlated with hepatic fibrosis (correlation coefficient > 0.8), and showed a similar expression pattern in NASH patients. Of these, two candidate genes (VWF and SERPINB9) encode secreted factors, warranting further investigations as potential biomarkers of human NASH progression. This study demonstrates key similarities in guinea pig and human NASH, supporting increased predictability when translating research findings to human patients.

Development of the Entorhinal Cortex Occurs via Parallel Lamination During Neurogenesis.

The entorhinal cortex (EC) is the spatial processing center of the brain and structurally is an interface between the three layered paleocortex and six layered neocortex, known as the periarchicortex. Limited studies indicate peculiarities in the formation of the EC such as early emergence of cells in layers (L) II and late deposition of LIII, as well as divergence in the timing of maturation of cell types in the superficial layers. In this study, we examine developmental events in the entorhinal cortex using an understudied model in neuroanatomy and development, the pig and supplement the research with BrdU labeling in the developing mouse EC. We determine the pig serves as an excellent anatomical model for studying human neurogenesis, given its long gestational length, presence of a moderate sized outer subventricular zone and early cessation of neurogenesis during gestation. Immunohistochemistry identified prominent clusters of OLIG2+ oligoprogenitor-like cells in the superficial layers of the lateral EC (LEC) that are sparser in the medial EC (MEC). These are first detected in the subplate during the early second trimester. MRI analyses reveal an acceleration of EC growth at the end of the second trimester. BrdU labeling of the developing MEC, shows the deeper layers form first and prior to the superficial layers, but the LV/VI emerges in parallel and the LII/III emerges later, but also in parallel. We coin this lamination pattern parallel lamination. The early born Reln+ stellate cells in the superficial layers express the classic LV marker, Bcl11b (Ctip2) and arise from a common progenitor that forms the late deep layer LV neurons. In summary, we characterize the developing EC in a novel animal model and outline in detail the formation of the EC. We further provide insight into how the periarchicortex forms in the brain, which differs remarkably to the inside-out lamination of the neocortex.

The canine activated platelet secretome (CAPS): A translational model of thrombin-evoked platelet activation response.

BACKGROUND: Domestic dogs represent a translational animal model to study naturally occurring human disease. Proteomics has emerged as a promising tool for characterizing human platelet pathophysiology; thus a detailed characterization of the core canine activated platelet secretome (CAPS) will enhance utilization of the canine model. The objectives of this study were development of a robust, high throughput, label-free approach for proteomic identification and quantification of the canine platelet (i) thrombin releasate proteins, and (ii) the protein subgroup that constitutes CAPS. METHODS: Platelets were isolated from 10 healthy dogs and stimulated with 50 nmol/L of γ-thrombin or saline. Proteins were in-solution trypsin-digested and analyzed by nano-liquid chromatography-tandem spectrometry. Core releasate proteins were defined as those present in 10 of 10 dogs, and CAPS defined as releasate proteins with a significantly higher abundance in stimulated versus saline controls (corrected P < .05). RESULTS: A total of 2865 proteins were identified; 1126 releasate proteins were present in all dogs, 650 were defined as CAPS. Among the differences from human platelets were a canine lack of platelet factor 4 and vascular endothelial growth factor C, and a 10- to 20-fold lower concentration of proteins such as haptoglobin, alpha-2 macroglobulin, von Willebrand factor, and amyloid-beta A4. Twenty-eight CAPS proteins, including cytokines, adhesion molecules, granule proteins, and calcium regulatory proteins have not previously been attributed to human platelets. CONCLUSIONS: CAPS proteins represent a robust characterization of a large animal platelet secretome and a novel tool to model platelet physiology, pathophysiology, and to identify translational biomarkers of platelet-mediated disease.

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species.

Multiple Sequence Alignments Enhance Boundary Definition of RNA Structures.

Self-contained structured domains of RNA sequences have often distinct molecular functions. Determining the boundaries of structured domains of a non-coding RNA (ncRNA) is needed for many ncRNA gene finder programs that predict RNA secondary structures in aligned genomes because these methods do not necessarily provide precise information about the boundaries or the location of the RNA structure inside the predicted ncRNA. Even without having a structure prediction, it is of interest to search for structured domains, such as for finding common RNA motifs in RNA-protein binding assays. The precise definition of the boundaries are essential for downstream analyses such as RNA structure modelling, e.g., through covariance models, and RNA structure clustering for the search of common motifs. Such efforts have so far been focused on single sequences, thus here we present a comparison for boundary definition between single sequence and multiple sequence alignments. We also present a novel approach, named RNAbound, for finding the boundaries that are based on probabilities of evolutionarily conserved base pairings. We tested the performance of two different methods on a limited number of Rfam families using the annotated structured RNA regions in the human genome and their multiple sequence alignments created from 14 species. The results show that multiple sequence alignments improve the boundary prediction for branched structures compared to single sequences independent of the chosen method. The actual performance of the two methods differs on single hairpin structures and branched structures. For the RNA families with branched structures, including transfer RNA (tRNA) and small nucleolar RNAs (snoRNAs), RNAbound improves the boundary predictions using multiple sequence alignments to median differences of -6 and -11.5 nucleotides (nts) for left and right boundary, respectively (window size of 200 nts).

Identification and characterization of novel conserved RNA structures in Drosophila.

BACKGROUND: Comparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). The increasing availability of related genomes now makes it possible to systematically search for compensatory base changes - and thus for conserved secondary structures - even in genomic regions that are poorly alignable in the primary sequence. The wealth of available transcriptome data can add valuable insight into expression and possible function for new ncRNA candidates. Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome. RESULTS: To search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, we conducted a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster and integrated this with an extensive set of tiling array data. The structural alignment screen revealed ∼30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, we found that most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units. CONCLUSIONS: This study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.

DotAligner: identification and clustering of RNA structure motifs.

The diversity of processed transcripts in eukaryotic genomes poses a challenge for the classification of their biological functions. Sparse sequence conservation in non-coding sequences and the unreliable nature of RNA structure predictions further exacerbate this conundrum. Here, we describe a computational method, DotAligner, for the unsupervised discovery and classification of homologous RNA structure motifs from a set of sequences of interest. Our approach outperforms comparable algorithms at clustering known RNA structure families, both in speed and accuracy. It identifies clusters of known and novel structure motifs from ENCODE immunoprecipitation data for 44 RNA-binding proteins.

Associating transcription factors and conserved RNA structures with gene regulation in the human brain.

Anatomical subdivisions of the human brain can be associated with different neuronal functions. This functional diversification is reflected by differences in gene expression. By analyzing post-mortem gene expression data from the Allen Brain Atlas, we investigated the impact of transcription factors (TF) and RNA secondary structures on the regulation of gene expression in the human brain. First, we modeled the expression of a gene as a linear combination of the expression of TFs. We devised an approach to select robust TF-gene interactions and to determine localized contributions to gene expression of TFs. Among the TFs with the most localized contributions, we identified EZH2 in the cerebellum, NR3C1 in the cerebral cortex and SRF in the basal forebrain. Our results suggest that EZH2 is involved in regulating ZIC2 and SHANK1 which have been linked to neurological diseases such as autism spectrum disorder. Second, we associated enriched regulatory elements inside differentially expressed mRNAs with RNA secondary structure motifs. We found a group of purine-uracil repeat RNA secondary structure motifs plus other motifs in neuron related genes such as ACSL4 and ERLIN2.

The identification and functional annotation of RNA structures conserved in vertebrates.

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.

RNAscClust: clustering RNA sequences using structure conservation and graph based motifs.

MOTIVATION: Clustering RNA sequences with common secondary structure is an essential step towards studying RNA function. Whereas structural RNA alignment strategies typically identify common structure for orthologous structured RNAs, clustering seeks to group paralogous RNAs based on structural similarities. However, existing approaches for clustering paralogous RNAs, do not take the compensatory base pair changes obtained from structure conservation in orthologous sequences into account. RESULTS: Here, we present RNAscClust , the implementation of a new algorithm to cluster a set of structured RNAs taking their respective structural conservation into account. For a set of multiple structural alignments of RNA sequences, each containing a paralog sequence included in a structural alignment of its orthologs, RNAscClust computes minimum free-energy structures for each sequence using conserved base pairs as prior information for the folding. The paralogs are then clustered using a graph kernel-based strategy, which identifies common structural features. We show that the clustering accuracy clearly benefits from an increasing degree of compensatory base pair changes in the alignments. AVAILABILITY AND IMPLEMENTATION: RNAscClust is available at http://www.bioinf.uni-freiburg.de/Software/RNAscClust . CONTACT: gorodkin@rth.dk or backofen@informatik.uni-freiburg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.