Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC.

The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes.

In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3'-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3'-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.

Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity and is frequently misregulated in human disease. Accordingly, an important goal of biomedical research is the development of tools capable of comprehensively, accurately, and efficiently profiling AS. Here, we describe Whippet, an easy-to-use RNA-seq analysis method that rapidly-with hardware requirements compatible with a laptop-models and quantifies AS events of any complexity without loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that one-third of human protein coding genes produce transcripts with complex AS events involving co-expression of two or more principal splice isoforms. We observe that high-entropy AS events are more prevalent in tumor relative to matched normal tissues and correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate future biomedical research.

Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.

We describe the application of a microarray platform, which combines information from exon body and splice-junction probes, to perform a quantitative analysis of tissue-specific alternative splicing (AS) for thousands of exons in mammalian cells. Through this system, we have analyzed global features of AS in major mouse tissues. The results provide numerous inferences for the functions of tissue-specific AS, insights into how the evolutionary history of exons can impact on their inclusion levels, and also information on how global regulatory properties of AS define tissue type. Like global transcription profiles, global AS profiles reflect tissue identity. Interestingly, we find that transcription and AS act independently on different sets of genes in order to define tissue-specific expression profiles. These results demonstrate the utility of our quantitative microarray platform and data for revealing important global regulatory features of AS.

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

Tight coordination of gene expression in the developing cerebellum is crucial for establishment of neuronal circuits governing motor and cognitive function. However, transcriptional changes alone do not explain all of the switches underlying neuronal differentiation. Here we unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. The motifs enriched in modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints by directly binding near the 3' splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, resulting in motor coordination defects, ataxia, and abnormal social behavior. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and establishes motor and cognitive circuitry in the developing cerebellum.

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is essential for viral replication, making it a promising target for antiviral drug and vaccine development. SARS-CoV-2 infected patients exhibit an uncoordinated immune response; however, the underlying mechanistic details of this imbalance remain obscure. Here, starting from a functional proteomics workflow, we cataloged the protein-protein interactions of SARS-CoV-2 proteins, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N localizes to stress granules and sequesters G3BPs away from their typical interaction partners, thus attenuating stress granule formation. We found that N binds directly to host mRNAs in cells, with a preference for 3' UTRs, and modulates target mRNA stability. We show that the N protein rewires the G3BP1 mRNA-binding profile and suppresses the physiological stress response of host cells, which may explain the imbalanced immune response observed in SARS-CoV-2 infected patients.

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control.

Roifman Syndrome is a rare congenital disorder characterized by growth retardation, cognitive delay, spondyloepiphyseal dysplasia and antibody deficiency. Here we utilize whole-genome sequencing of Roifman Syndrome patients to reveal compound heterozygous rare variants that disrupt highly conserved positions of the RNU4ATAC small nuclear RNA gene, a minor spliceosome component that is essential for minor intron splicing. Targeted sequencing confirms allele segregation in six cases from four unrelated families. RNU4ATAC rare variants have been recently reported to cause microcephalic osteodysplastic primordial dwarfism, type I (MOPD1), whose phenotype is distinct from Roifman Syndrome. Strikingly, all six of the Roifman Syndrome cases have one variant that overlaps MOPD1-implicated structural elements, while the other variant overlaps a highly conserved structural element not previously implicated in disease. RNA-seq analysis confirms extensive and specific defects of minor intron splicing. Available allele frequency data suggest that recessive genetic disorders caused by RNU4ATAC rare variants may be more prevalent than previously reported.

Eukaryotic cells are defined by compartments through which the trafficking of macromolecules is mediated by large complexes, such as the nuclear pore, transport vesicles and intraflagellar transport. The assembly and maintenance of these complexes is facilitated by endomembrane coatomers, long suspected to be divergently related on the basis of structural and more recently phylogenomic analysis. By performing supervised walks in sequence space across coatomer superfamilies, we uncover subtle sequence patterns that have remained elusive to date, ultimately unifying eukaryotic coatomers by divergent evolution. The conserved residues shared by 3,502 endomembrane coatomer components are mapped onto the solenoid superhelix of nucleoporin and COPII protein structures, thus determining the invariant elements of coatomer architecture. This ancient structural motif can be considered as a universal signature connecting eukaryotic coatomers involved in multiple cellular processes across cell physiology and human disease.

Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

The evolution of metazoans from their unicellular ancestors was one of the most important events in the history of life. However, the cellular and genetic changes that ultimately led to the evolution of multicellularity are not known. In this study, we describe an aggregative multicellular stage in the protist Capsaspora owczarzaki, a close unicellular relative of metazoans. Remarkably, transition to the aggregative stage is associated with significant upregulation of orthologs of genes known to establish multicellularity and tissue architecture in metazoans. We further observe transitions in regulated alternative splicing during the C. owczarzaki life cycle, including the deployment of an exon network associated with signaling, a feature of splicing regulation so far only observed in metazoans. Our results reveal the existence of a highly regulated aggregative stage in C. owczarzaki and further suggest that features of aggregative behavior in an ancestral protist may had been co-opted to develop some multicellular properties currently seen in metazoans. DOI: http://dx.doi.org/10.7554/eLife.01287.001.

Transcriptome profiling of patterns of RNA expression is a powerful approach to identify networks of genes that play a role in disease. To date, most mRNA profiling of tissues has been accomplished using microarrays, but next-generation sequencing can offer a richer and more comprehensive picture.

There is growing evidence for the involvement of Y-complex nucleoporins (Y-Nups) in cellular processes beyond the inner core of nuclear pores of eukaryotes. To comprehensively assess the range of possible functions of Y-Nups, we delimit their structural and functional properties by high-specificity sequence profiles and tissue-specific expression patterns. Our analysis establishes the presence of Y-Nups across eukaryotes with novel composite domain architectures, supporting new moonlighting functions in DNA repair, RNA processing, signaling and mitotic control. Y-Nups associated with a select subset of the discovered domains are found to be under tight coordinated regulation across diverse human and mouse cell types and tissues, strongly implying that they function in conjunction with the nuclear pore. Collectively, our results unearth an expanded network of Y-Nup interactions, thus supporting the emerging view of the Y-complex as a dynamic protein assembly with diverse functional roles in the cell.

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.

Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.