Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Coordination of Alternative Splicing and Alternative Polyadenylation revealed by Targeted Long-Read Sequencing.

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to resolve the connectivity of alternative exons to alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Investigating the Streptococcus sinensis competence regulon through a combination of transcriptome analysis and phenotypic evaluation.

Streptococcus sinensis is a recently identified member of the Mitis group of streptococci. This species has been associated with infective endocarditis; however its mechanisms of pathogenesis and virulence are not fully understood. This study aimed to investigate the influence of the competence-stimulating peptide (CSP) and the competence regulon quorum-sensing circuitry (ComABCDE) on subsequent gene transcription and expression, as well as resultant phenotypes. In this study we confirmed the native CSP identity, ascertained when endogenous CSP was produced and completed a transcriptome-wide analysis of all genes following CSP exposure. RNA sequencing analysis revealed the upregulation of genes known to be associated with competence, biofilm formation and virulence. As such, a variety of phenotypic assays were utilized to assess the correlation between increased mRNA expression and potential phenotype response, ultimately gaining insight into the effects of CSP on both gene expression and developed phenotypes. The results indicated that the addition of exogenous CSP aided in competence development and successful transformation, yielding an average transformation efficiency comparable to that of other Mitis group streptococci. Additional studies are needed to further delineate the effects of CSP exposure on biofilm formation and virulence. Overall, this study provides novel information regarding S. sinensis and provides a substantial foundation on which this species and its role in disease pathogenesis can be further investigated.

Loss of circRNAs from the crh-1 gene extends the mean lifespan in Caenorhabditis elegans.

Accumulation of circular RNAs (circRNAs) during aging occurs on a genome-wide level for multiple organisms, but its significance is unknown. Generating circRNA loss-of-function mutants is difficult because the vast majority of these RNAs are comprised of exons shared with protein-coding mRNAs. In Caenorhabditis elegans, most circRNAs were previously found to accumulate during aging. Two of the most abundant, age-accumulating circRNAs are generated from exon 4 of the crh-1 gene (circ-crh-1). Here, we found that the biogenesis of circ-crh-1 was regulated by the double-stranded RNA-binding protein ADR-1. We identified Reverse Complementary Match (RCM) sequences in introns flanking circ-crh-1. Using CRISPR-Cas9, we deleted the downstream RCM and found that this completely eliminated expression of the circRNA without affecting linear mRNA expression from the crh-1 gene. Remarkably, worms lacking circ-crh-1 exhibited a significantly longer mean lifespan. Lifespan was partially restored to wild type by expression of circ-crh-1 in neural tissues. Widespread transcriptome alterations in circ-crh-1 mutants were identified using RNA-Seq. Moving forward, intronic RCM deletion using CRISPR should be a widely applicable method to identify lifespan-regulating circRNAs in C. elegans.

NOVA2 regulates neural circRNA biogenesis.

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establishes a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circEfnb2 was impaired when YCAY clusters located in flanking introns were mutagenized. CLIP (cross-linking and immunoprecipitation) and additional reporter analyses demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain.

CRISPR-Mediated Knockout of Long 3' UTR mRNA Isoforms in mESC-Derived Neurons.

Alternative cleavage and polyadenylation (APA) is pervasive, occurring for more than 70% of human and mouse genes. Distal poly(A) site selection to generate longer 3' UTR mRNA isoforms is prevalent in the nervous system, affecting thousands of genes. Here, we establish mouse embryonic stem cell (mESC)-derived neurons (mES-neurons) as a suitable system to study long 3' UTR isoforms. RNA-seq analysis revealed that mES-neurons show widespread 3' UTR lengthening that closely resembles APA patterns found in mouse cortex. mESCs are highly amenable to genetic manipulation. We present a method to eliminate long 3' UTR isoform expression using CRISPR/Cas9 editing. This approach can lead to clones with the desired deletion within several weeks. We demonstrate this strategy on the Mprip gene as a proof-of-principle. To confirm loss of long 3' UTR expression and the absence of cryptic poly(A) site usage stemming from the CRISPR deletion, we present a simple and cost-efficient targeted long-read RNA-sequencing strategy using the Oxford Nanopore Technologies platform. Using this method, we confirmed specific loss of the Mprip long 3' UTR isoform. CRISPR gene editing of mESCs thus serves as a highly relevant platform for studying the molecular and cellular functions of long 3' UTR mRNA isoforms.

Overlapping Activities of ELAV/Hu Family RNA Binding Proteins Specify the Extended Neuronal 3' UTR Landscape in Drosophila.

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.

Elimination of Calm1 long 3'-UTR mRNA isoform by CRISPR-Cas9 gene editing impairs dorsal root ganglion development and hippocampal neuron activation in mice.

The majority of mouse and human genes are subject to alternative cleavage and polyadenylation (APA), which most often leads to the expression of two or more alternative length 3' untranslated region (3'-UTR) mRNA isoforms. In neural tissues, there is enhanced expression of APA isoforms with longer 3'-UTRs on a global scale, but the physiological relevance of these alternative 3'-UTR isoforms is poorly understood. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that generates short (Calm1-S) and long (Calm1-L) 3'-UTR mRNA isoforms via APA. We found Calm1-L expression to be largely restricted to neural tissues in mice including the dorsal root ganglion (DRG) and hippocampus, whereas Calm1-S was more broadly expressed. smFISH revealed that both Calm1-S and Calm1-L were subcellularly localized to neural processes of primary hippocampal neurons. In contrast, cultured DRG showed restriction of Calm1-L to soma. To investigate the in vivo functions of Calm1-L, we implemented a CRISPR-Cas9 gene editing strategy to delete a small region encompassing the Calm1 distal poly(A) site. This eliminated Calm1-L expression while maintaining expression of Calm1-S Mice lacking Calm1-L (Calm1ΔL/ΔL ) exhibited disorganized DRG migration in embryos, and reduced experience-induced neuronal activation in the adult hippocampus. These data indicate that Calm1-L plays functional roles in the central and peripheral nervous systems.

Emerging Roles for 3' UTRs in Neurons.

The 3' untranslated regions (3' UTRs) of mRNAs serve as hubs for post-transcriptional control as the targets of microRNAs (miRNAs) and RNA-binding proteins (RBPs). Sequences in 3' UTRs confer alterations in mRNA stability, direct mRNA localization to subcellular regions, and impart translational control. Thousands of mRNAs are localized to subcellular compartments in neurons-including axons, dendrites, and synapses-where they are thought to undergo local translation. Despite an established role for 3' UTR sequences in imparting mRNA localization in neurons, the specific RNA sequences and structural features at play remain poorly understood. The nervous system selectively expresses longer 3' UTR isoforms via alternative polyadenylation (APA). The regulation of APA in neurons and the neuronal functions of longer 3' UTR mRNA isoforms are starting to be uncovered. Surprising roles for 3' UTRs are emerging beyond the regulation of protein synthesis and include roles as RBP delivery scaffolds and regulators of alternative splicing. Evidence is also emerging that 3' UTRs can be cleaved, leading to stable, isolated 3' UTR fragments which are of unknown function. Mutations in 3' UTRs are implicated in several neurological disorders-more studies are needed to uncover how these mutations impact gene regulation and what is their relationship to disease severity.

Elav-Mediated Exon Skipping and Alternative Polyadenylation of the Dscam1 Gene Are Required for Axon Outgrowth.

Many metazoan genes express alternative long 3' UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3' UTR isoforms because of alternative polyadenylation (APA). Here, we found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3' UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3' UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon outgrowth in Drosophila. The Dscam1 long 3' UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing.

CircRNA accumulation: A new hallmark of aging?

Circular RNAs (circRNAs) are a newly appreciated class of RNAs found across phyla that are generated most commonly from back-splicing of protein-coding exons. Recent profiling of circRNAs genome-wide has shown that hundreds of circRNAs dramatically increase in expression during aging in the brains of multiple organisms. No other class of transcripts has been found to show such a strong correlation with aging as circRNAs-could they be playing a role in the aging process? Here, we discuss the different methods used to profile circRNAs and discuss current limitations of these approaches. We argue that age-related increases in global circRNA levels likely result from their high stability. The functions of circRNAs are only beginning to emerge, and it is an open question whether circRNA accumulation impacts the aging brain. We discuss experimental approaches that could illuminate whether age-accumulation of circRNAs are detrimental or protective to the aging brain.

Age-related defects in short-term plasticity are reversed by acetyl-L-carnitine at the mouse calyx of Held.

Hearing acuity and sound localization are affected by aging and may contribute to cognitive dementias. Although loss of sensorineural conduction is well documented to occur with age, little is known regarding short-term synaptic plasticity in central auditory nuclei. Age-related changes in synaptic transmission properties were evaluated at the mouse calyx of Held, a sign-inverting relay synapse in the circuit for sound localization, in juvenile adults (1 month old) and late middle-aged (18-21 months old) mice. Synaptic timing and short-term plasticity were severely disrupted in older mice. Surprisingly, acetyl-l-carnitine (ALCAR), an anti-inflammatory agent that facilitates mitochondrial function, fully reversed synaptic transmission delays and defects in short-term plasticity in aged mice to reflect transmission similar to that seen in juvenile adults. These findings support ALCAR supplementation as an adjuvant to improve short-term plasticity and potentially central nervous system performance in animals compromised by age and/or neurodegenerative disease.

Genome-Wide circRNA Profiling from RNA-seq Data.

The genome-wide expression patterns of circular RNAs (circRNAs) are of increasing interest for their potential roles in normal cellular homeostasis, development, and disease. Thousands of circRNAs have been annotated from various species in recent years. Analysis of publically available or user-generated rRNA-depleted total RNA-seq data can be performed to uncover new circRNA expression trends. Here we provide a primer for profiling circRNAs from RNA-seq datasets. The description is tailored for the wet lab scientist with limited or no experience in analyzing RNA-seq data. We begin by describing how to access and interpret circRNA annotations. Next, we cover converting circRNA annotations into junction sequences that are used as scaffolds to align RNA-seq reads. Lastly, we visit quantifying circRNA expression trends from the alignment data.

Global accumulation of circRNAs during aging in Caenorhabditis elegans.

BACKGROUND: Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5' and 3' ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues. RESULTS: Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated a high-confidence set of 1166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by RT-qPCR. The expression of 459 circRNAs was determined to be distinct from the expression of linear RNAs from the same host genes, demonstrating host gene independence of circRNA age-accumulation. CONCLUSIONS: We attribute the global scale of circRNA age-accumulation to the high composition of post-mitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine whether there are functional consequences of circRNA accumulation during aging.

Transcriptome profiling of aging Drosophila photoreceptors reveals gene expression trends that correlate with visual senescence.

BACKGROUND: Aging is associated with functional decline of neurons and increased incidence of both neurodegenerative and ocular disease. Photoreceptor neurons in Drosophila melanogaster provide a powerful model for studying the molecular changes involved in functional senescence of neurons since decreased visual behavior precedes retinal degeneration. Here, we sought to identify gene expression changes and the genomic features of differentially regulated genes in photoreceptors that contribute to visual senescence. RESULTS: To identify gene expression changes that could lead to visual senescence, we characterized the aging transcriptome of Drosophila sensory neurons highly enriched for photoreceptors. We profiled the nuclear transcriptome of genetically-labeled photoreceptors over a 40 day time course and identified increased expression of genes involved in stress and DNA damage response, and decreased expression of genes required for neuronal function. We further show that combinations of promoter motifs robustly identify age-regulated genes, suggesting that transcription factors are important in driving expression changes in aging photoreceptors. However, long, highly expressed and heavily spliced genes are also more likely to be downregulated with age, indicating that other mechanisms could contribute to expression changes at these genes. Lastly, we identify that circular RNAs (circRNAs) strongly increase during aging in photoreceptors. CONCLUSIONS: Overall, we identified changes in gene expression in aging Drosophila photoreceptors that could account for visual senescence. Further, we show that genomic features predict these age-related changes, suggesting potential mechanisms that could be targeted to slow the rate of age-associated visual decline.

Loss of adult skeletal muscle stem cells drives age-related neuromuscular junction degeneration.

Neuromuscular junction degeneration is a prominent aspect of sarcopenia, the age-associated loss of skeletal muscle integrity. Previously, we showed that muscle stem cells activate and contribute to mouse neuromuscular junction regeneration in response to denervation (Liu et al., 2015). Here, we examined gene expression profiles and neuromuscular junction integrity in aged mouse muscles, and unexpectedly found limited denervation despite a high level of degenerated neuromuscular junctions. Instead, degenerated neuromuscular junctions were associated with reduced contribution from muscle stem cells. Indeed, muscle stem cell depletion was sufficient to induce neuromuscular junction degeneration at a younger age. Conversely, prevention of muscle stem cell and derived myonuclei loss was associated with attenuation of age-related neuromuscular junction degeneration, muscle atrophy, and the promotion of aged muscle force generation. Our observations demonstrate that deficiencies in muscle stem cell fate and post-synaptic myogenesis provide a cellular basis for age-related neuromuscular junction degeneration and associated skeletal muscle decline.

Genome-wide profiling of the 3' ends of polyadenylated RNAs.

Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.

Emerging Functions of Circular RNAs.

Many thousands of Circular RNAs (circRNAs) have recently been identified in metazoan genomes by transcriptome-wide sequencing. Most circRNAs are generated by back-splicing events from exons of protein-coding genes. A great deal of progress has recently been made in understanding the genome-wide expression patterns, biogenesis, and regulation of circRNAs. To date, however, few functions of circRNAs have been identified. CircRNAs are preferentially expressed in neural tissues and some are found at synapses, suggesting possible functions in the nervous system. Several circRNAs have been shown to function as microRNA "sponges" to counteract microRNA mediated repression of mRNA. New functions for circRNAs are arising, including protein sequestration, transcriptional regulation, and potential functions in cancer. Here, we highlight the recent progress made in understanding the biogenesis and regulation of circRNAs, discuss newly uncovered circRNA functions, and explain the methodological approaches that could reveal more exciting and unexpected roles for these RNAs.

CircRNA accumulation in the aging mouse brain.

Circular RNAs (circRNAs) are a newly appreciated class of RNAs expressed across diverse phyla. These enigmatic transcripts are most commonly generated by back-splicing events from exons of protein-coding genes. This results in highly stable RNAs due to the lack of free 5' and 3' ends. CircRNAs are enriched in neural tissues, suggesting that they might have neural functions. Here, we sought to determine whether circRNA accumulation occurs during aging in mice. Total RNA-seq profiling of young (1 month old) and aged (22 month old) cortex, hippocampus and heart samples was performed. This led to the confident detection of 6,791 distinct circRNAs across these samples, including 675 novel circRNAs. Analysis uncovered a strong bias for circRNA upregulation during aging in neural tissues. These age-accumulation trends were verified for individual circRNAs by RT-qPCR and Northern analysis. In contrast, comparison of aged versus young hearts failed to reveal a global trend for circRNA upregulation. Age-accumulation of circRNAs in brain tissues was found to be largely independent from linear RNA expression of host genes. These findings suggest that circRNAs might play biological roles relevant to the aging nervous system.

IsoSCM: improved and alternative 3' UTR annotation using multiple change-point inference.

Major applications of RNA-seq data include studies of how the transcriptome is modulated at the levels of gene expression and RNA processing, and how these events are related to cellular identity, environmental condition, and/or disease status. While many excellent tools have been developed to analyze RNA-seq data, these generally have limited efficacy for annotating 3' UTRs. Existing assembly strategies often fragment long 3' UTRs, and importantly, none of the algorithms in popular use can apportion data into tandem 3' UTR isoforms, which are frequently generated by alternative cleavage and polyadenylation (APA). Consequently, it is often not possible to identify patterns of differential APA using existing assembly tools. To address these limitations, we present a new method for transcript assembly, Isoform Structural Change Model (IsoSCM) that incorporates change-point analysis to improve the 3' UTR annotation process. Through evaluation on simulated and genuine data sets, we demonstrate that IsoSCM annotates 3' termini with higher sensitivity and specificity than can be achieved with existing methods. We highlight the utility of IsoSCM by demonstrating its ability to recover known patterns of tissue-regulated APA. IsoSCM will facilitate future efforts for 3' UTR annotation and genome-wide studies of the breadth, regulation, and roles of APA leveraging RNA-seq data. The IsoSCM software and source code are available from our website https://github.com/shenkers/isoscm.