Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition.

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, limitations, and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for continuous extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies, while the containers and reproducible workflows could easily be deployed and extended to evaluate new methods or data sets.

The X-linked splicing regulator MBNL3 has been co-opted to restrict placental growth in eutherians.

Understanding the regulatory interactions that control gene expression during the development of novel tissues is a key goal of evolutionary developmental biology. Here, we show that Mbnl3 has undergone a striking process of evolutionary specialization in eutherian mammals resulting in the emergence of a novel placental function for the gene. Mbnl3 belongs to a family of RNA-binding proteins whose members regulate multiple aspects of RNA metabolism. We find that, in eutherians, while both Mbnl3 and its paralog Mbnl2 are strongly expressed in placenta, Mbnl3 expression has been lost from nonplacental tissues in association with the evolution of a novel promoter. Moreover, Mbnl3 has undergone accelerated protein sequence evolution leading to changes in its RNA-binding specificities and cellular localization. While Mbnl2 and Mbnl3 share partially redundant roles in regulating alternative splicing, polyadenylation site usage and, in turn, placenta maturation, Mbnl3 has also acquired novel biological functions. Specifically, Mbnl3 knockout (M3KO) alone results in increased placental growth associated with higher Myc expression. Furthermore, Mbnl3 loss increases fetal resource allocation during limiting conditions, suggesting that location of Mbnl3 on the X chromosome has led to its role in limiting placental growth, favoring the maternal side of the parental genetic conflict.

Analysis of RNA Polyadenylation in Healthy and Osteoarthritic Human Articular Cartilage.

Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3' regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3' RNA sequencing approach, where each read runs 3' to 5' from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.

A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells.

BACKGROUND: Temperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. RESULTS: The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14-29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. CONCLUSION: Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range.

CELF4 regulates translation and local abundance of a vast set of mRNAs, including genes associated with regulation of synaptic function.

RNA-binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA-binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%-20% of the transcriptome, with striking specificity for the mRNA 3' untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Na(v)1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA-binding proteins play in regulating and shaping the activity of neural circuits.

Analysis of alternative splicing associated with aging and neurodegeneration in the human brain.

Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.

iCLIP predicts the dual splicing effects of TIA-RNA interactions.

The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5' splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5' splice sites. Surprisingly, TIA binding at 5' splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3' splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5' splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.

SNPsyn: detection and exploration of SNP-SNP interactions.

SNPsyn (http://snpsyn.biolab.si) is an interactive software tool for the discovery of synergistic pairs of single nucleotide polymorphisms (SNPs) from large genome-wide case-control association studies (GWAS) data on complex diseases. Synergy among SNPs is estimated using an information-theoretic approach called interaction analysis. SNPsyn is both a stand-alone C++/Flash application and a web server. The computationally intensive part is implemented in C++ and can run in parallel on a dedicated cluster or grid. The graphical user interface is written in Adobe Flash Builder 4 and can run in most web browsers or as a stand-alone application. The SNPsyn web server hosts the Flash application, receives GWAS data submissions, invokes the interaction analysis and serves result files. The user can explore details on identified synergistic pairs of SNPs, perform gene set enrichment analysis and interact with the constructed SNP synergy network.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

dictyExpress: a Dictyostelium discoideum gene expression database with an explorative data analysis web-based interface.

BACKGROUND: Bioinformatics often leverages on recent advancements in computer science to support biologists in their scientific discovery process. Such efforts include the development of easy-to-use web interfaces to biomedical databases. Recent advancements in interactive web technologies require us to rethink the standard submit-and-wait paradigm, and craft bioinformatics web applications that share analytical and interactive power with their desktop relatives, while retaining simplicity and availability. RESULTS: We have developed dictyExpress, a web application that features a graphical, highly interactive explorative interface to our database that consists of more than 1000 Dictyostelium discoideum gene expression experiments. In dictyExpress, the user can select experiments and genes, perform gene clustering, view gene expression profiles across time, view gene co-expression networks, perform analyses of Gene Ontology term enrichment, and simultaneously display expression profiles for a selected gene in various experiments. Most importantly, these tasks are achieved through web applications whose components are seamlessly interlinked and immediately respond to events triggered by the user, thus providing a powerful explorative data analysis environment. CONCLUSION: dictyExpress is a precursor for a new generation of web-based bioinformatics applications with simple but powerful interactive interfaces that resemble that of the modern desktop. While dictyExpress serves mainly the Dictyostelium research community, it is relatively easy to adapt it to other datasets. We propose that the design ideas behind dictyExpress will influence the development of similar applications for other model organisms.

High-Resolution RNA Maps Suggest Common Principles of Splicing and Polyadenylation Regulation by TDP-43.

Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3' mRNA sequencing. This reveals at nucleotide resolution the "RNA maps" describing how the RNA binding positions of RBPs relate to their regulatory functions. We use this approach to examine how TDP-43, an RBP involved in several neurodegenerative diseases, binds around its regulated poly(A) sites. Binding close to the poly(A) site generally represses, whereas binding further downstream enhances use of the site, which is similar to TDP-43 binding around regulated exons. Our RNAmotifs2 software also identifies sequence motifs that cluster together with the binding motifs of TDP-43. We conclude that TDP-43 directly regulates diverse types of pre-mRNA processing according to common position-dependent principles.

RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing.

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.

Transcriptional profiling of Dictyostelium with RNA sequencing.

Transcriptional profiling methods have been utilized in the analysis of various biological processes in Dictyostelium. Recent advances in high-throughput sequencing have increased the resolution and the dynamic range of transcriptional profiling. Here we describe the utility of RNA sequencing with the Illumina technology for production of transcriptional profiles. We also describe methods for data mapping and storage as well as common and specialized tools for data analysis, both online and offline.

Bacterial discrimination by dictyostelid amoebae reveals the complexity of ancient interspecies interactions.

BACKGROUND: Amoebae and bacteria interact within predator-prey and host-pathogen relationships, but the general response of amoeba to bacteria is not well understood. The amoeba Dictyostelium discoideum feeds on, and is colonized by, diverse bacterial species, including Gram-positive [Gram(+)] and Gram-negative [Gram(-)] bacteria, two major groups of bacteria that differ in structure and macromolecular composition. RESULTS: Transcriptional profiling of D. discoideum revealed sets of genes whose expression is enriched in amoebae interacting with different species of bacteria, including sets that appear specific to amoebae interacting with Gram(+) or with Gram(-) bacteria. In a genetic screen utilizing the growth of mutant amoebae on a variety of bacteria as a phenotypic readout, we identified amoebal genes that are only required for growth on Gram(+) bacteria, including one that encodes the cell-surface protein gp130, as well as several genes that are only required for growth on Gram(-) bacteria, including one that encodes a putative lysozyme, AlyL. These genes are required for parts of the transcriptional response of wild-type amoebae, and this allowed their classification into potential response pathways. CONCLUSIONS: We have defined genes that are critical for amoebal survival during feeding on Gram(+), or Gram(-), bacteria that we propose form part of a regulatory network that allows D. discoideum to elicit specific cellular responses to different species of bacteria in order to optimize survival.

Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain.

Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.

iCLIP--transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs. Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP). These approaches were prone to identifying indirect or non-physiological interactions. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq). Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking. Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1). Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation.

Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.

In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance, however, it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hnRNP particles determines their effect on the inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.