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SummaryThe 3’ untranslated region (3’UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5’ ends of mRNAs, additionally reveals a large number of apparently 5’ capped RNAs derived from 3’UTRs. Here we provide the first direct evidence that these 3’UTR-derived RNAs are indeed capped and often more abundant than the corresponding full-length mRNAs. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA knockdowns, we find that these 3’UTR-derived RNAs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form RNA-G-quadruplexes and are enriched by RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validates several 3’UTR-derived RNAs, demonstrates their abundance and shows that they tend not to co-localise with the parental mRNAs. Taken together, we provide new insights into the origin and abundance of 3’UTR-derived RNAs, show the utility of CAGE-seq for their quantitative detection, and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia (FTD), with patients sometimes experiencing elements of both conditions (ALS-FTSD). For mutations associated with ALS-FTSD, such as theC9orf72hexanucleotide repeat expansion (HRE), the factors influencing where an individual may lie on this spectrum require further characterisation. Here, using NanoString molecular barcoding with a panel of 770 neuroinflammatory genes, we interrogate inflammatory dysregulation at the level of gene expression. We identified 20 dysregulated neuroinflammatory genes in the motor cortex of deeply clinically phenotyped C9-ALSpost-mortemcases, with enrichment of microglial and inflammatory response gene sets. Our analyses also revealed two distinct ALS-related neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. Two genes with significant correlations to available clinical metrics were selected for validation:FKBP5andBDNF. FKBP5 and its signalling partner, NF-κB, appeared to have a cell-type-specific staining distribution, with activated (i.e., nuclear) NF-κB immunoreactivity in C9-ALS. Expression ofBDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™in situhybridisation. Finally, we compared NPS between C9-ALS cases and those from deeply clinically phenotyped sporadic ALS (sALS) and SOD1-ALS cohorts, with NPS1 and NPS2 appearing across all cohorts. A subset of these signatures was also detected in publicly available RNA-sequencing data from independent C9-ALS and sALS cohorts, underscoring the relevance of these pathways across cohorts. Our findings highlight the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within genetic and sporadic cohorts.

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.

Clinical heterogeneity observed across patients with amyotrophic lateral sclerosis (ALS) is a known complicating factor in identifying potential therapeutics, even within cohorts with the same mutation, such as C9orf72 hexanucleotide repeat expansions (HREs). Thus, further understanding of pathways underlying this heterogeneity is essential for appropriate ALS trial stratification and the meaningful assessment of clinical outcomes. It has been shown that both inflammation and protein misfolding can influence ALS pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. However, there has yet to be a systematic and quantitative assessment of immunohistochemical markers to interrogate the potential relevance of these pathways in an unbiased manner. To investigate this, we extensively characterised features of commonly used glial activation and protein misfolding stains in thousands of images of post-mortem tissue from a heterogeneous cohort of deeply clinically profiled patients with a C9orf72 HRE. Using a random forest model, we show that microglial staining features are the most accurate classifiers of disease status in our panel and that clinicopathological relationships exist between microglial activation status, TDP-43 pathology, and language dysfunction. Furthermore, we detected spatially resolved changes in fused in sarcoma (FUS) staining, suggesting that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Interestingly, no one feature alone significantly impacted the predictiveness of the model, indicating that the collective examination of all features, or a combination of several features, is what allows the model to be predictive. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9-ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological relationships within and between cohorts. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Single-cell RNA-sequencing (scRNA-seq) has emerged in recent years as a breakthrough technology to understand RNA metabolism at cellular resolution. In addition to allowing new cell types and states to be identified, scRNA-seq can permit cell-type specific differential gene expression changes, pre-mRNA processing events, gene regulatory networks and single-cell developmental trajectories to be uncovered. More recently, a new wave of multi-omic adaptations and complementary spatial transcriptomics workflows have been developed that facilitate the collection of even more holistic information from individual cells. These developments have unprecedented potential to provide penetrating new insights into the basic neural cell dynamics and molecular mechanisms relevant to the nervous system in both health and disease. In this review we discuss this maturation of single-cell RNA-sequencing over the past decade, and review the different adaptations of the technology that can now be applied both at different scales and for different purposes. We conclude by highlighting how these methods have already led to many exciting discoveries across neuroscience that have furthered our cellular understanding of the neurological disease.

Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are regarded as two ends of a pathogenetic spectrum, termed ALS-frontotemporal spectrum disorder (ALS-FTSD). However, it is currently difficult to predict where on the spectrum an individual will lie, especially for patients with C9orf72 hexanucleotide repeat expansions (HRE), a mutation associated with both ALS and FTD. It has been shown that both inflammation and protein misfolding influence aspects of ALS and ALS-FTSD disease pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. Previous studies have highlighted markers which may influence C9orf72-associated disease presentation in a targeted fashion, though there has yet to be a systematic and quantitative assessment of common immunohistochemical markers to investigate the significance of these pathways in an unbiased manner. Here we report the first extensive digital pathological assessment with random forest modelling of pathological markers often used in neuropathology practice. This study profiles glial activation and protein misfolding in a cohort of deeply clinically profiled post-mortem tissue from patients with a C9orf72 HRE, who either met the criteria for a diagnosis of ALS or ALS-FTSD. We show that microglial immunohistochemical staining features, both morphological and spatial, are the best independent classifiers of disease status and that clinicopathological associations exist between microglial activation status and cognitive dysfunction in ALS-FTSD patients with C9orf72 HRE. Furthermore, we show that spatially resolved changes in FUS staining are also an accurate predictor of disease status, implying that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9orf72 ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological associations between cohorts.

Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

Single cell transcriptome profiling has emerged as a breakthrough technology for the high-resolution understanding of complex cellular systems. Here we report a flexible, cost-effective and user-friendly droplet-based microfluidics system, called the Nadia Instrument, that can allow 3′ mRNA capture of ~ 50,000 single cells or individual nuclei in a single run. The precise pressure-based system demonstrates highly reproducible droplet size, low doublet rates and high mRNA capture efficiencies that compare favorably in the field. Moreover, when combined with the Nadia Innovate, the system can be transformed into an adaptable setup that enables use of different buffers and barcoded bead configurations to facilitate diverse applications. Finally, by 3′ mRNA profiling asynchronous human and mouse cells at different phases of the cell cycle, we demonstrate the system's ability to readily distinguish distinct cell populations and infer underlying transcriptional regulatory networks. Notably this provided supportive evidence for multiple transcription factors that had little or no known link to the cell cycle (e.g. DRAP1, ZKSCAN1 and CEBPZ). In summary, the Nadia platform represents a promising and flexible technology for future transcriptomic studies, and other related applications, at cell resolution.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0749-3) contains supplementary material, which is available to authorized users.

SummarySingle cell transcriptome profiling has emerged as a breakthrough technology for the high-resolution understanding of complex cellular systems. Here we report a flexible, cost-effective and user-friendly droplet-based microfluidics system, called the Nadia Instrument, that can allow 3’ mRNA capture of ∼50,000 single cells or individual nuclei in a single run. The precise pressure-based system demonstrates highly reproducible droplet size, low doublet rates and high mRNA capture efficiencies that compare favorably in the field. Moreover, when combined with the Nadia Innovate, the system can be transformed into an adaptable setup that enables use of different buffers and barcoded bead configurations to facilitate diverse applications. Finally, by 3’ mRNA profiling asynchronous human and mouse cells at different phases of the cell cycle, we demonstrate the system’s ability to readily distinguish distinct cell populations and infer underlying transcriptional regulatory networks. Notably this identified multiple transcription factors that had little or no known link to the cell cycle (e.g. DRAP1, ZKSCAN1 and CEBPZ). In summary, the Nadia platform represents a promising and flexible technology for future transcriptomic studies, and other related applications, at cell resolution.

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

SUMMARYCellular DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill-defined. Given the centrality of RNA polymerase II (Pol II) promoter-proximal pause release in transcriptional control, we evaluated its importance in DDR. Here we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates Pol II elongation and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb). This is mediated by genotoxic stress-enhanced binding of RBM7 to 7SK snRNA (7SK), the scaffold of the 7SK small nuclear ribonucleoprotein (7SK snRNP) which inhibits P-TEFb. In turn, P-TEFb relocates from 7SK snRNP to chromatin to induce transcription of short units including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with RBM7 or P-TEFb provokes cellular hypersensitivity to DNA damage-inducing agents through activation of apoptotic program. By alleviating the inhibition of P-TEFb, RBM7 thus facilitates Pol II elongation to enable a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult. Our work uncovers a new paradigm in stress-dependent control of Pol II pause release, and offers the promise for designing novel anti-cancer interventions using RBM7 and P-TEFb antagonists in combination with DNA-damaging chemotherapeutics.

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map human spliceosome engagement with snRNAs and pre-mRNAs. This identified over 50,000 branchpoints (BPs) that have canonical sequence and structural features. Moreover, it revealed 7 binding peaks around BPs and splice sites, each precisely overlapping with binding profiles of specific splicing factors. We show how the binding patterns of these RBPs are affected by the position and strength of BPs. For example, strong or proximally located BPs preferentially bind SF3 rather than U2AF complex. Notably, these effects are partly neutralized during spliceosomal assembly in a way that depends on the core spliceosomal protein PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

It is generally believed that splicing removes introns as single units from pre-mRNA transcripts. However, some long D. melanogaster introns contain a cryptic site, called a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing1,2. The extent to which recursive splicing occurs in other species and its mechanistic basis remain unclear. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of a “RS-exon” that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform due to competition with a reconstituted 5′ splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic exons or promoters that are prevalent in long introns, but which fail to reconstitute an efficient 5′ splice site. Most RS-exons contain a premature stop codon such that their inclusion may decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling inclusion of cryptic elements with RS-exons.

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries.

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries.

Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3′ fragment capture and degradome sequencing. This reveals that endogenous transcripts can have NMD-eliciting features at various positions, including upstream open reading frames (uORFs), premature termination codons (PTCs), and long 3′ UTRs. We find that NMD substrates with PTCs undergo constitutive SMG6-dependent endocleavage, rather than SMG7-dependent exonucleolytic decay. In contrast, the turnover of NMD substrates containing uORFs and long 3′ UTRs involves both SMG6- and SMG7-dependent endo- and exonucleolytic decay, respectively. This suggests that the extent to which SMG6 and SMG7 degrade NMD substrates is determined by the mRNA architecture.

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, whilst their perturbed activity can contribute to disease aetiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent crosslinks formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct crosslink sites in the high throughput sequenced cDNA libraries.

The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases. This article is part of a Special Issue entitled: RNA and splicing regulation in neurodegeneration.

Schizophrenia and the affective disorders, here comprising bipolar disorder and major depressive disorder, are psychiatric illnesses that lead to significant morbidity and mortality worldwide. Whilst understanding of their pathobiology remains limited, large case-control studies have recently identified single nucleotide polymorphisms (SNPs) associated with these disorders. However, discerning the functional effects of these SNPs has been difficult as the associated causal genes are unknown. Here we evaluated whether schizophrenia and affective disorder associated-SNPs are correlated with gene expression within human brain tissue. Specifically, to identify expression quantitative trait loci (eQTLs), we leveraged disorder-associated SNPs identified from six Psychiatric Genomics Consortium and CONVERGE Consortium studies with gene expression levels in post-mortem, neurologically-normal tissue from two independent human brain tissue expression datasets (UK Brain Expression Consortium (UKBEC) and Genotype-Tissue Expression (GTEx)). We identified 6 188 and 16 720 cis-acting SNPs exceeding genome-wide significance (p−8) in the UKBEC and GTEx datasets, respectively. 1 288 cis-eQTLs were significant in a metaanalysis leveraging overlapping brain regions and were associated with expression of 15 genes, including three non-coding RNAs. One cis-eQTL, rs 16969968, results in a functionally disruptive missense mutation in CHRNA5, a schizophrenia-implicated gene. Meta-analysis identified 297 trans-eQTLs associated with 24 genes that were significant in a region-specific manner. Importantly, comparing across tissues, we find that blood eQTLs largely do not capture brain cis-eQTLs. This study identifies putatively causal genes whose expression in region-specific brain tissue may contribute to the risk of schizophrenia and affective disorders.

RNA interference (RNAi) represents a powerful post-transcriptional gene silencing network which fine-tunes gene expression in all eukaryotic cells. The endogenous triggers of RNAi, microRNAs (miRNAs), are proposed to regulate expression of up to a third of all protein-coding genes, and have been shown to have critical roles in developmental processes including in the central nervous system and skeletal muscle. Further, many have been reported to display differential expression in various disease states. Here we describe present understanding of the biogenesis and function of miRNAs, review current knowledge of miRNA abnormalities in both human neurological and skeletal muscle disease and discuss their potential as novel disease biomarkers. Finally, we highlight the many ways in which the miRNA pathway may be targeted for therapeutic benefit. © 2011 Springer-Verlag.

Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies.

Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon–intron boundaries and the 3′ UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, the release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.

Post-transcriptional gene silencing (PTGS) via RNA interference (RNAi) is a vital gene regulatory mechanism for fine-tuning gene expression. RNAi effectors termed microRNAs (miRNAs) are implicated in various aspects of animal development and normal physiological function, while dysregulation has been linked to several pathologies. Several atypical miRNA biogenesis pathways have been identified, yet in most cases the reasons for their emergence remain unclear. One of these atypical pathways is the mirtron pathway, where short introns are excised by splicing to generate intermediates of the RNAi pathway, with no cleavage by the microprocessor. Closely related pathways involving tailed-mirtron and simtron biogenesis have also been described. There is extensive evidence that mirtrons function as miRNAs, and while some are evolutionarily conserved across similar species, others appear to have emerged relatively recently. In addition, through exploitation of the potent and sequence-specific silencing capabilities of RNAi, synthetic mirtrons may have potential for overcoming certain therapeutic challenges. © 2012 John Wiley & Sons, Ltd.

Post-transcriptional regulation plays a major role in the generation of cell type diversity. In particular, alternative splicing increases diversification of transcriptome between tissues, in different cell types within a tissue, and even in different compartments of the same cell. The complexity of alternative splicing has increased during evolution. With increasing sophistication, however, comes greater potential for malfunction of these intricate processes. Indeed, recent years have uncovered a wealth of disease-causing mutations affecting RNA-binding proteins and non-coding regions on RNAs, highlighting the importance of studying disease mechanisms that act at the level of RNA processing. For instance, mutations in TARDBP and FUS, or a repeat expansion in the intronic region of the C9ORF72 gene, can all cause amyotrophic lateral sclerosis. We discuss how interspecies differences highlight the necessity for human model systems to complement existing non-human approaches to study neurodegenerative disorders. We conclude by discussing the improvements that could further increase the promise of human pluripotent stem for cell-based disease modeling. This article is part of a Special Issue entitled “RNA-Binding Proteins”.

The past decade has seen intense scientific interest in non-coding RNAs. In particular, the discovery and subsequent exploitation of gene silencing via RNA interference (RNAi) has revolutionized the way in which gene expression is now studied and understood. It is now well established that post-transcriptional gene silencing (PTGS) by the microRNA (miRNA) and other RNAi-associated pathways represents an essential layer of complexity to gene regulation. Gene silencing using RNAi additionally demonstrates huge potential as a therapeutic strategy for eliminating pathogenic gene expression. Yet despite the early promise and excitement of gene-specific silencing, several critical hurdles remain to be overcome before widespread clinical adoption. These include off-target effects, toxicity due to saturation of the endogenous RNAi functions, limited duration of silencing, and effective targeted delivery. In recent years, a range of novel strategies for producing RNA-mediated silencing have been developed that can circumvent many of these hurdles, including small internally segmented interfering RNAs, tandem hairpin RNAs, and pri-miRNA cluster mimics. This review discusses RNA-mediated silencing in light of this recent research, and highlights the benefits and limitations conferred by these novel gene-silencing strategies.

Alternative splicing is universally accredited for expanding the information encoded within the transcriptome. In recent years, several tightly regulated alternative splicing events have been reported which do not lead to generation of protein products, but lead to unstable mRNA isoforms. Instead these transcripts are targets for NMD (nonsense-mediated decay) or retained in the nucleus and degraded. In the present review I discuss the regulation of these events, and how many have been implicated in control of gene expression that is instrumental to a number of developmental paradigms. I further discuss their relevance to disease settings and conclude by highlighting technologies that will aid identification of more candidate events in future.

RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.

Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3′ UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial mirtrons cloned as eGFP introns. Deep sequencing of artificial mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies.

Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 mirtron and incorporated these into artificial mirtrons targeting Parkinson's disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial mirtrons make this an attractive approach for future RNAi applications. © 2012 The Author(s).

Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5-10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a 'walk-through' series of RNA Pol III-expressed shRNAs targeting both the (Alpha)-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the (Alpha)-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5'RACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future.

Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT-PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.

Purification of proteins cross-linked to mRNAs has identified 800 mRNA-binding proteins and their characteristics.