Genome-wide kinetic profiling of pre-mRNA 3' end cleavage.

Cleavage and polyadenylation is necessary for the formation of mature mRNA molecules. The rate at which this process occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. Despite advances in high-throughput approaches for global kinetic profiling of RNA maturation, genome-wide 3' end cleavage rates have never been measured. Here, we describe a novel approach to estimate the rates of cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. We find that 3' end cleavage is fast on average, with half-lives under a minute, but highly variable across individual sites. Rapid cleavage is promoted by the presence of canonical sequence elements and an increased density of polyadenylation signals near a cleavage site. Finally, we find that cleavage rates are associated with the localization of RNA polymerase II at the end of a gene, and faster cleavage leads to quicker degradation of downstream readthrough RNA. Our findings shed light on the features important for efficient 3' end cleavage and the regulation of transcription termination.

mRNA initiation and termination are spatially coordinated.

The expression of a precise mRNA transcriptome is crucial for establishing cell identity and function, with dozens of alternative isoforms produced for a single gene sequence. The regulation of mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processing mechanisms across a gene. Decisions involved in mRNA initiation and termination underlie the largest extent of mRNA isoform diversity, but little is known about any relationships between decisions at both ends of mRNA molecules. Here, we systematically profile the joint usage of mRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues and species. Using both short and long read RNA-seq data, we observe that mRNAs preferentially using upstream TSSs also tend to use upstream PASs, and congruently, the usage of downstream sites is similarly paired. This observation suggests that mRNA 5' end choice may directly influence mRNA 3' ends. Our results suggest a novel "Positional Initiation-Termination Axis" (PITA), in which the usage of alternative terminal sites are coupled based on the order in which they appear in the genome. PITA isoforms are more likely to encode alternative protein domains and use conserved sites. PITA is strongly associated with the length of genomic features, such that PITA is enriched in longer genes with more area devoted to regions that regulate alternative 5' or 3' ends. Strikingly, we found that PITA genes are more likely than non-PITA genes to have multiple, overlapping chromatin structural domains related to pairing of ordinally coupled start and end sites. In turn, PITA coupling is also associated with fast RNA Polymerase II (RNAPII) trafficking across these long gene regions. Our findings indicate that a combination of spatial and kinetic mechanisms couple transcription initiation and mRNA 3' end decisions based on ordinal position to define the expression mRNA isoforms.

Challenges in identifying mRNA transcript starts and ends from long-read sequencing data.

Long-read sequencing (LRS) technologies have the potential to revolutionize scientific discoveries in RNA biology, especially by enabling the comprehensive identification and quantification of full length mRNA isoforms. However, inherently high error rates make the analysis of long-read sequencing data challenging. While these error rates have been characterized for sequence and splice site identification, it is still unclear how accurately LRS reads represent transcript start and end sites. Here, we systematically assess the variability and accuracy of mRNA terminal ends identified by LRS reads across multiple sequencing platforms. We find substantial inconsistencies in both the start and end coordinates of LRS reads spanning a gene, such that LRS reads often fail to accurately recapitulate annotated or empirically derived terminal ends of mRNA molecules. To address this challenge, we introduce an approach to condition reads based on empirically derived terminal ends and identified a subset of reads that are more likely to represent full-length transcripts. Our approach can improve transcriptome analyses by enhancing the fidelity of transcript terminal end identification, but may result in lower power to quantify genes or discover novel isoforms. Thus, it is necessary to be cautious when selecting sequencing approaches and/or interpreting data from long-read RNA sequencing.

A cohesin traffic pattern genetically linked to gene regulation.

Cohesin-mediated loop extrusion has been shown to be blocked at specific cis-elements, including CTCF sites, producing patterns of loops and domain boundaries along chromosomes. Here we explore such cis-elements, and their role in gene regulation. We find that transcription termination sites of active genes form cohesin- and RNA polymerase II-dependent domain boundaries that do not accumulate cohesin. At these sites, cohesin is first stalled and then rapidly unloaded. Start sites of transcriptionally active genes form cohesin-bound boundaries, as shown before, but are cohesin-independent. Together with cohesin loading, possibly at enhancers, these sites create a pattern of cohesin traffic that guides enhancer-promoter interactions. Disrupting this traffic pattern, by removing CTCF, renders cells sensitive to knockout of genes involved in transcription initiation, such as the SAGA complexes, and RNA processing such DEAD/H-Box RNA helicases. Without CTCF, these factors are less efficiently recruited to active promoters.

G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.

High-throughput analysis of ANRIL circRNA isoforms in human pancreatic islets.

The antisense non-coding RNA in the INK locus (ANRIL) is a hotspot for genetic variants associated with cardiometabolic disease. We recently found increased ANRIL abundance in human pancreatic islets from donors with certain Type II Diabetes (T2D) risk-SNPs, including a T2D risk-SNP located within ANRIL exon 2 associated with beta cell proliferation. Recent studies have found that expression of circular species of ANRIL is linked to the regulation of cardiovascular phenotypes. Less is known about how the abundance of circular ANRIL may influence T2D phenotypes. Herein, we sequence circular RNA in pancreatic islets to characterize circular isoforms of ANRIL. We identify several consistently expressed circular ANRIL isoforms whose expression is correlated across dozens of individuals and characterize ANRIL splice sites that are commonly involved in back-splicing. We find that samples with the T2D risk allele in ANRIL exon 2 had higher ratios of circular to linear ANRIL compared to protective-allele carriers, and that higher circular:linear ANRIL was associated with decreased beta cell proliferation. Our study points to a combined involvement of both linear and circular ANRIL species in T2D phenotypes and opens the door for future studies of the molecular mechanisms by which ANRIL impacts cellular function in pancreatic islets.

Widespread occurrence of hybrid internal-terminal exons in human transcriptomes.

Messenger RNA isoform differences are predominantly driven by alternative first, internal, and last exons. Despite the importance of classifying exons to understand isoform structure, few tools examine isoform-specific exon usage. We recently observed that alternative transcription start sites often arise near internal exons, often creating "hybrid" first/internal exons. To systematically detect hybrid exons, we built the hybrid-internal-terminal (HIT) pipeline to classify exons depending on their isoform-specific usage. On the basis of splice junction reads in RNA sequencing data and probabilistic modeling, the HIT index identified thousands of previously misclassified hybrid first-internal and internal-last exons. Hybrid exons are enriched in long genes and genes involved in RNA splicing and have longer flanking introns and strong splice sites. Their usage varies considerably across human tissues. By developing the first method to classify exons according to isoform contexts, our findings document the occurrence of hybrid exons, a common quirk of the human transcriptome.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system.

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

Environmental influences on RNA processing: Biochemical, molecular and genetic regulators of cellular response.

RNA processing has emerged as a key mechanistic step in the regulation of the cellular response to environmental perturbation. Recent work has uncovered extensive remodeling of transcriptome composition upon environmental perturbation and linked the impacts of this molecular plasticity to health and disease outcomes. These isoform changes and their underlying mechanisms are varied-involving alternative sites of transcription initiation, alternative splicing, and alternative cleavage at the 3' end of the mRNA. The mechanisms and consequences of differential RNA processing have been characterized across a range of common environmental insults, including chemical stimuli, immune stimuli, heat stress, and cancer pathogenesis. In each case, there are perturbation-specific contributions of local (cis) regulatory elements or global (trans) factors and downstream consequences. Overall, it is clear that choices in isoform usage involve a balance between the usage of specific genetic elements (i.e., splice sites, polyadenylation sites) and the timing at which certain decisions are made (i.e., transcription elongation rate). Fine-tuned cellular responses to environmental perturbation are often dependent on the genetic makeup of the cell. Genetic analyses of interindividual variation in splicing have identified genetic effects on splicing that contribute to variation in complex traits. Finally, the increase in the number of tissue types and environmental conditions analyzed for RNA processing is paralleled by the need to develop appropriate analytical tools. The combination of large datasets, novel methods and conditions explored promises to provide a much greater understanding of the role of RNA processing response in human phenotypic variation. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin.

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.

Numerous recursive sites contribute to accuracy of splicing in long introns in flies.

Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.

Bayesian nonparametric discovery of isoforms and individual specific quantification.

Most human protein-coding genes can be transcribed into multiple distinct mRNA isoforms. These alternative splicing patterns encourage molecular diversity, and dysregulation of isoform expression plays an important role in disease etiology. However, isoforms are difficult to characterize from short-read RNA-seq data because they share identical subsequences and occur in different frequencies across tissues and samples. Here, we develop BIISQ, a Bayesian nonparametric model for isoform discovery and individual specific quantification from short-read RNA-seq data. BIISQ does not require isoform reference sequences but instead estimates an isoform catalog shared across samples. We use stochastic variational inference for efficient posterior estimates and demonstrate superior precision and recall for simulations compared to state-of-the-art isoform reconstruction methods. BIISQ shows the most gains for low abundance isoforms, with 36% more isoforms correctly inferred at low coverage versus a multi-sample method and 170% more versus single-sample methods. We estimate isoforms in the GEUVADIS RNA-seq data and validate inferred isoforms by associating genetic variants with isoform ratios.

The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture.

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning ('intron definition') or exon-spanning ('exon definition') pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60-70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.

Environmental perturbations lead to extensive directional shifts in RNA processing.

Environmental perturbations have large effects on both organismal and cellular traits, including gene expression, but the extent to which the environment affects RNA processing remains largely uncharacterized. Recent studies have identified a large number of genetic variants associated with variation in RNA processing that also have an important role in complex traits; yet we do not know in which contexts the different underlying isoforms are used. Here, we comprehensively characterized changes in RNA processing events across 89 environments in five human cell types and identified 15,300 event shifts (FDR = 15%) comprised of eight event types in over 4,000 genes. Many of these changes occur consistently in the same direction across conditions, indicative of global regulation by trans factors. Accordingly, we demonstrate that environmental modulation of splicing factor binding predicts shifts in intron retention, and that binding of transcription factors predicts shifts in alternative first exon (AFE) usage in response to specific treatments. We validated the mechanism hypothesized for AFE in two independent datasets. Using ATAC-seq, we found altered binding of 64 factors in response to selenium at sites of AFE shift, including ELF2 and other factors in the ETS family. We also performed AFE QTL mapping in 373 individuals and found an enrichment for SNPs predicted to disrupt binding of the ELF2 factor. Together, these results demonstrate that RNA processing is dramatically changed in response to environmental perturbations through specific mechanisms regulated by trans factors.

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection.

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3' UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3' UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3' UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.

Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters.

Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here we found that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but owing to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.

The genetic and mechanistic basis for variation in gene regulation.

It is now well established that noncoding regulatory variants play a central role in the genetics of common diseases and in evolution. However, until recently, we have known little about the mechanisms by which most regulatory variants act. For instance, what types of functional elements in DNA, RNA, or proteins are most often affected by regulatory variants? Which stages of gene regulation are typically altered? How can we predict which variants are most likely to impact regulation in a given cell type? Recent studies, in many cases using quantitative trait loci (QTL)-mapping approaches in cell lines or tissue samples, have provided us with considerable insight into the properties of genetic loci that have regulatory roles. Such studies have uncovered novel biochemical regulatory interactions and led to the identification of previously unrecognized regulatory mechanisms. We have learned that genetic variation is often directly associated with variation in regulatory activities (namely, we can map regulatory QTLs, not just expression QTLs [eQTLs]), and we have taken the first steps towards understanding the causal order of regulatory events (for example, the role of pioneer transcription factors). Yet, in most cases, we still do not know how to interpret overlapping combinations of regulatory interactions, and we are still far from being able to predict how variation in regulatory mechanisms is propagated through a chain of interactions to eventually result in changes in gene expression profiles.

Comparative studies of gene regulatory mechanisms.

It has become increasingly clear that changes in gene regulation have played an important role in adaptive evolution both between and within species. Over the past five years, comparative studies have moved beyond simple characterizations of differences in gene expression levels within and between species to studying variation in regulatory mechanisms. We still know relatively little about the precise chain of events that lead to most regulatory adaptations, but we have taken significant steps towards understanding the relative importance of changes in different mechanisms of gene regulatory evolution. In this review, we first discuss insights from comparative studies in model organisms, where the available experimental toolkit is extensive. We then focus on a few recent comparative studies in primates, where the limited feasibility of experimental manipulation dictates the approaches that can be used to study gene regulatory evolution.