TDP-43 condensation properties specify its RNA binding and regulation

Summary: Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These “binding-region condensates” are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution

Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB.

Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3.We thank Nejc Haberman (UCL) for assisting in the generation of the splicing maps and preparation of the iCLIP data. This work was supported by Wellcome Trust programme grants to CWJS (077877 and 092900), and by grants to EE and NB BIO2011-23920 and RNAREG (CSD2009-00080) from the Spanish Government and by the Sandra Ibarra Foundation for Cancer (FSI2013). JA was supported by a Boehringer Ingelheim Fonds studentship.This is the final version of the article. It first appeared from EMBO Press/Wiley via http://dx.doi.org/10.15252/embj.20148985

Evolutionarily conserved human targets of adenosine to inosine RNA editing

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice

Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5' splice site strength.

BACKGROUND: Sequential assembly of the human spliceosome on RNA transcripts regulates splicing across the human transcriptome. The core spliceosome component PRPF8 is essential for spliceosome assembly through its participation in ribonucleoprotein (RNP) complexes for splice-site recognition, branch-point formation and catalysis. PRPF8 deficiency is linked to human diseases like retinitis pigmentosa or myeloid neoplasia, but its genome-wide effects on constitutive and alternative splicing remain unclear. RESULTS: Here, we show that alterations in RNA splicing patterns across the human transcriptome that occur in conditions of restricted cellular PRPF8 abundance are defined by the altered splicing of introns with weak 5' splice sites. iCLIP of spliceosome components reveals that PRPF8 depletion decreases RNP complex formation at most splice sites in exon-intron junctions throughout the genome. However, impaired splicing affects only a subset of human transcripts, enriched for mitotic cell cycle factors, leading to mitotic arrest. Preferentially retained introns and differentially used exons in the affected genes contain weak 5' splice sites, but are otherwise indistinguishable from adjacent spliced introns. Experimental enhancement of splice-site strength in mini-gene constructs overcomes the effects of PRPF8 depletion on the kinetics and fidelity of splicing during transcription. CONCLUSIONS: Competition for PRPF8 availability alters the transcription-coupled splicing of RNAs in which weak 5' splice sites predominate, enabling diversification of human gene expression during biological processes like mitosis. Our findings exemplify the regulatory potential of changes in the core spliceosome machinery, which may be relevant to slow-onset human genetic diseases linked to PRPF8 deficiency

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences

Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

© 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society. There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotidebased biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications

MPromDb update 2010: an integrated resource for annotation and visualization of mammalian gene promoters and ChIP-seq experimental data

MPromDb (Mammalian Promoter Database) is a curated database that strives to annotate gene promoters identified from ChIP-seq results with the goal of providing an integrated resource for mammalian transcriptional regulation and epigenetics. We analyzed 507 million uniquely aligned RNAP-II ChIP-seq reads from 26 different data sets that include six human cell-types and 10 distinct mouse cell/tissues. The updated MPromDb version consists of computationally predicted (novel) and known active RNAP-II promoters (42 893 human and 48 366 mouse promoters) from various data sets freely available at NCBI GEO database. We found that 36% and 40% of protein-coding genes have alternative promoters in human and mouse genomes and ∼40% of promoters are tissue/cell specific. The identified RNAP-II promoters were annotated using various known and novel gene models. Additionally, for novel promoters we looked into other evidences—GenBank mRNAs, spliced ESTs, CAGE promoter tags and mRNA-seq reads. Users can search the database based on gene id/symbol, or by specific tissue/cell type and filter results based on any combination of tissue/cell specificity, Known/Novel, CpG/NonCpG, and protein-coding/non-coding gene promoters. We have also integrated GBrowse genome browser with MPromDb for visualization of ChIP-seq profiles and to display the annotations. The current release of MPromDb can be accessed at http://bioinformatics.wistar.upenn.edu/MPromDb/

Secondary structure is required for 3′ splice site recognition in yeast

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3′ splice site (3′ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3′ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3′ss distance and masked potential 3′ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3′ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3′ss recognition

Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR)

Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2)/endothelial cell-specific chemotaxis regulator (ECSCR). In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2), differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293). Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF)-induced cell migration. Taken together, we have provided several lines of experimental evidence that two mouse ECSCR splicing variants/isoform precursors exist. They are differentially expressed in a variety of tissue types and likely involved in modulation of vascular EC migration. We have also defined the gene structure of mouse ECSCR using bioinformatics tools, which provides new information towards a better understanding of alternative splicing of ECSCR

A structural determinant required for RNA editing

RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing