Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders.

A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.

Gomafu lncRNA knockout mice exhibit mild hyperactivity with enhanced responsiveness to the psychostimulant methamphetamine.

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.

Malat1 is not an essential component of nuclear speckles in mice.

Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine- and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA--Neat1--which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions.

Long non-coding RNAs in nuclear bodies.

High-throughput analyses of mammalian transcriptomes have revealed that more than half of the transcripts produced by RNA polymerase II are non-protein-coding. One class of these non-coding transcripts is the long non-coding RNAs (lncRNAs), which are more than 200 nucleotides in length and are molecularly indistinguishable from other protein-coding mRNAs. Although the molecular functions of these lncRNAs have long remained unknown, emerging evidence implicates the functional involvement of lncRNAs in the regulation of gene expression through the modification of chromatin, maintenance of subnuclear structures, transport of specific mRNAs, and control of pre-mRNA splicing. Here, we discuss the functions of a distinct group of vertebrate-specific lncRNAs, NEAT1/MENε/ß/VINC, MALAT1/NEAT2, and Gomafu/RNCR2/MIAT, which accumulate abundantly within the nucleus as RNA components of specific nuclear bodies.

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation.

The rate of RNA polymerase II (Pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing (AS) is not well understood. We performed experiments to perturb Pol II elongation and then globally compared AS patterns with genome-wide Pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with Pol II elongation inhibition-dependent changes in AS. Under conditions that interfere with Pol II elongation, including cell stress, increased Pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, AS, and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels.

Functional coordination of alternative splicing in the mammalian central nervous system.

BACKGROUND: Alternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood. RESULTS: Using quantitative AS microarray profiling, we have identified a large number of widely expressed mouse genes that contain single or coordinated pairs of alternative exons that are spliced in a tissue regulated fashion. The majority of these AS events display differential regulation in central nervous system (CNS) tissues. Approximately half of the corresponding genes have neural specific functions and operate in common processes and interconnected pathways. Differential regulation of AS in the CNS tissues correlates strongly with a set of mostly new motifs that are predominantly located in the intron and constitutive exon sequences neighboring CNS-regulated alternative exons. Different subsets of these motifs are correlated with either increased inclusion or increased exclusion of alternative exons in CNS tissues, relative to the other profiled tissues. CONCLUSION: Our findings provide new evidence that specific cellular processes in the mammalian CNS are coordinated at the level of AS, and that a complex splicing code underlies CNS specific AS regulation. This code appears to comprise many new motifs, some of which are located in the constitutive exons neighboring regulated alternative exons. These data provide a basis for understanding the molecular mechanisms by which the tissue specific functions of widely expressed genes are coordinated at the level of AS.

Identification and characterization of RED120: a conserved PWI domain protein with links to splicing and 3'-end formation.

Precursor (pre)-mRNA splicing can impact the efficiency of coupled steps in gene expression. SRm160 (SR-related nuclear matrix protein of 160 kDa), is a splicing coactivator that also functions as a 3'-end cleavage-stimulatory factor. Here, we have identified an evolutionary-conserved SRm160-interacting protein, referred to as hRED120 (for human Arg/Glu/Asp-rich protein of 120 kDa). hRED120 contains a conventional RNA recognition motif and, like SRm160, a PWI nucleic acid binding domain, suggesting that it has the potential to bridge different RNP complexes. Also, similar to SRm160, hRED120 associates with snRNP components, and remains associated with mRNA after splicing. Simultaneous suppression in Caenorhabditis elegans of the ortholog of hRED120 with the orthologs of splicing and 3'-end processing factors results in aberrant growth or developmental defects. These results suggest that RED120 may function to couple splicing with mRNA 3'-end formation.

Global analysis of alternative splicing during T-cell activation.

The role of alternative splicing (AS) in eliciting immune responses is poorly understood. We used quantitative AS microarray profiling to survey changes in AS during activation of Jurkat cells, a leukemia-derived T-cell line. Our results indicate that approximately 10-15% of the profiled alternative exons undergo a >10% change in inclusion level during activation. The majority of the genes displaying differential AS levels are distinct from the set of genes displaying differential transcript levels. These two gene sets also have overlapping yet distinct functional roles. For example, genes that show differential AS patterns during T-cell activation are often closely associated with cell-cycle regulation, whereas genes with differential transcript levels are highly enriched in functions associated more directly with immune defense and cytoskeletal architecture. Previously unknown AS events were detected in genes that have important roles in T-cell activation, and these AS level changes were also observed during the activation of normal human peripheral CD4+ and CD8+ lymphocytes. In summary, by using AS microarray profiling, we have discovered many new AS changes associated with T-cell activation. Our results suggest an extensive role for AS in the regulation of the mammalian immune response.

Technologies for the global discovery and analysis of alternative splicing.

During the past approximately 20 years, studies on alternative splicing (AS) have largely been directed at the identification and characterization of factors and mecha nisms responsible for the control of splice site selection, using model substrates and on a case by case basis. These studies have provided a wealth of information on the factors and interactions that control formation of the spliceosome. However, relatively little is known about the global regulatory properties of AS. Important questions that need to be addressed are: which exons are alternatively spliced and under which cellular contexts, what are the functional roles of AS events in different cellular contexts, and how are AS events controlled and coordinated with each other and with other levels of gene regulation to achieve cell- and development-specific functions. During the past several years, new technologies and experimental strategies have provided insight into these questions. For example, custom microarrays and data analysis tools are playing a prominent role in the discovery and analysis of splicing regulation. Moreover, several non-microarray-based technologies are emerging that will likely further fuel progress in this area. This review focuses on recent advances made in the development and application of high-throughput methods to study AS.

Role for PSF in mediating transcriptional activator-dependent stimulation of pre-mRNA processing in vivo.

In a recent study, we provided evidence that strong promoter-bound transcriptional activators result in higher levels of splicing and 3'-end cleavage of nascent pre-mRNA than do weak promoter-bound activators and that this effect of strong activators requires the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II). In the present study, we have investigated the mechanism of activator- and CTD-mediated stimulation of pre-mRNA processing. Affinity chromatography experiments reveal that two factors previously implicated in the coupling of transcription and pre-mRNA processing, PSF and p54(nrb)/NonO, preferentially bind a strong rather than a weak activation domain. Elevated expression in human 293 cells of PSF bypasses the requirement for a strong activator to promote efficient splicing and 3'-end cleavage. Truncation of the pol II CTD, which consists of 52 repeats of the consensus heptapeptide sequence YSPTSPS, to 15 heptapeptide repeats prevents PSF-dependent stimulation of splicing and 3'-end cleavage. Moreover, PSF and p54(nrb)/NonO bind in vitro to the wild-type CTD but not to the truncated 15-repeat CTD, and domains in PSF that are required for binding to activators and to the CTD are also important for the stimulation of pre-mRNA processing. Interestingly, activator- and CTD-dependent stimulation of splicing mediated by PSF appears to primarily affect the removal of first introns. Collectively, these results suggest that the recruitment of PSF to activated promoters and the pol II CTD provides a mechanism by which transcription and pre-mRNA processing are coordinated within the cell.