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1.  Depolarization and CaM Kinase IV Modulate NMDA Receptor Splicing through Two Essential RNA Elements 
PLoS Biology  2007;5(2):e40.
Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.
Alternative splicing of NMDA receptor 1 exon 21 is reversibly repressed by depolarization in a CaMK IV-dependent manner in neurons. This suggests splicing is finely tuned by dynamic activity inputs.
Author Summary
Multiple mechanisms direct changes in neuronal activity in response to external stimuli, ranging from short-acting modifications of membrane proteins to longer-acting changes in gene expression. A frequently regulated step in gene expression is the pre-mRNA splicing reaction in which the inclusion of exons (protein-coding sequences) or the position of splice sites produces alternatively spliced mRNA isoforms encoding functionally different proteins. Here, we study splicing of the NMDA receptor, which responds to the neurotransmitter glutamate to modify neuronal activity. We show that the splicing of an important exon (E21) in the NMDA receptor subunit NR1 mRNA is repressed by cell depolarization and activation of the intracellular signaling molecule, CaMK IV. We find that this splicing repression is mediated by two regulatory sequences within the exon itself. One sequence is similar to a previously described regulatory element that had not been known to function in an exon. The other is a new element. The characterization of these elements as a family of degenerate sequences allowed the identification of a group of exons sharing responsiveness to cell depolarization and CamK IV. These results define a new set of gene expression changes that may occur in modulating neuronal activity.
doi:10.1371/journal.pbio.0050040
PMCID: PMC1790950  PMID: 17298178
2.  Genome-wide analysis of light-regulated alternative splicing mediated by photoreceptors in Physcomitrella patens 
Genome Biology  2014;15(1):R10.
Background
Light is one of the most important factors regulating plant growth and development. Light-sensing photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although many levels of gene expression are modulated by photoreceptors, regulation at the mRNA splicing step remains unclear.
Results
We performed high-throughput mRNA sequencing to analyze light-responsive changes in alternative splicing in the moss Physcomitrella patens, and found that a large number of alternative splicing events were induced by light in the moss protonema. Light-responsive intron retention preferentially occurred in transcripts involved in photosynthesis and translation. Many of the alternatively spliced transcripts were expressed from genes with a function relating to splicing or light signaling, suggesting a potential impact on pre-mRNA splicing and photomorphogenic gene regulation in response to light. Moreover, most light-regulated intron retention was induced immediately upon light exposure, while motif analysis identified a repetitive GAA motif that may function as an exonic regulatory cis element in light-mediated alternative splicing. Further analysis in gene-disrupted mutants was consistent with a function for multiple red-light photoreceptors in the upstream regulation of light-responsive alternative splicing.
Conclusions
Our results indicate that intensive alternative splicing occurs in non-vascular plants and that, during photomorphogenesis, light regulates alternative splicing with transcript selectivity. We further suggest that alternative splicing is rapidly fine-tuned by light to modulate gene expression and reorganize metabolic processes, and that pre-mRNA cis elements are involved in photoreceptor-mediated splicing regulation.
doi:10.1186/gb-2014-15-1-r10
PMCID: PMC4054894  PMID: 24398233
3.  Differential effects of PKA-controlled CaMKK2 variants on neuronal differentiation 
RNA Biology  2011;8(6):1061-1072.
Regulation between protein kinases is critical for the establishment of signaling pathways/networks to orchestrate cellular processes. Besides posttranslational phosphorylation, alternative pre-mRNA splicing is another way to control kinase properties, but splicing regulation between two kinases and the effect of resulting variants on cells have not been explored. We examined the effect of the protein kinase A (PKA) pathway on the alternative splicing and variant properties of the Ca++/calmodulin-dependent protein kinase kinase 2 (CaMKK2) gene in B35 neuroblastoma cells. Inclusion of the exon 16 of CaMKK2 was significantly reduced by H89, a PKA selective inhibitor. Consistently, overexpressed PKA strongly promoted the exon inclusion in a CaMKK2 sequence-dependent way in splicing reporter assays. In vitro, purified CaMKK2 variant proteins were kinase-active. In cells, they were differentially phosphorylated by PKA. In RNA interference assays, CaMKK2 was required for forskolin-induced neurite growth. Interestingly, overexpression of the variant without exon 16 (−E16) promoted neurite elongation while the other one (+E16) promoted neurite branching; in contrast, reduction of the latter variant enhanced neurite elongation. Moreover, the variants are differentially expressed and the exon 16-containing transcripts highly enriched in the brain, particularly the cerebellum and hippocampus. Thus, PKA regulates the alternative splicing of CaMKK2 to produce variants that differentially modulate neuronal differentiation. Taken together with the many distinct variants of kinases, alternative splicing regulation likely adds another layer of modulation between protein kinases in cellular signaling networks.
doi:10.4161/rna.8.6.16691
PMCID: PMC3256423  PMID: 21957496
alternative splicing; protein kinase A; cross-regulation; neuronal differentiation; phosphorylation
4.  Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo 
Journal of Virology  2000;74(13):5902-5910.
Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.
PMCID: PMC112086  PMID: 10846071
5.  Exon Silencing by UAGG Motifs in Response to Neuronal Excitation 
PLoS Biology  2007;5(2):e36.
Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.
Exon skipping of NMDA R1 receptor exon 21 (CI cassette) is mediated by UAGG-type silencing motifs, presenting a mechanism by which external stimuli communicate to the nuclear splicing machinery in neurons.
Author Summary
The modular features of a protein's architecture are regulated after transcription by the process of alternative pre-mRNA splicing. Conditions that excite or stress neurons can induce changes in some splicing patterns, suggesting that cellular pathways can take advantage of the flexibility of splicing to tune their protein activities for adaptation or survival. Although the phenomenon of the inducible splicing switch (or inducible exon) is well documented, the molecular underpinnings of these curious changes have remained mysterious.
We describe methods to study how the glutamate NMDA receptor, which is a fundamental component of interneuronal signaling and plasticity, undergoes an inducible switch in its splicing pattern in primary neurons. This splicing switch promotes the skipping of an exon that encodes the CI cassette protein module, which is thought to communicate signals from the membrane to the cell nucleus during neuronal activity. We show that this induced splicing event is regulated in neurons by a three-part (UAGG-type) sequence code for exon silencing, and demonstrate a wider role for exon-skipping responsiveness in transcripts with known synaptic functions that also harbor a similar sequence code.
doi:10.1371/journal.pbio.0050036
PMCID: PMC1790952  PMID: 17298175
6.  Kinetic competition during the transcription cycle results in stochastic RNA processing 
eLife  2014;3:e03939.
Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release.
DOI: http://dx.doi.org/10.7554/eLife.03939.001
eLife digest
To make a protein, part of a DNA sequence is copied to make a messenger RNA (or mRNA) molecule in a process known as transcription. The enzyme that builds an mRNA molecule first binds to a start point on a DNA strand, and then uses the DNA sequence to build a ‘pre-mRNA’ molecule until a stop signal is reached.
To make the final mRNA molecule, sections called introns are removed from the pre-mRNA molecules, and the parts left behind—known as exons—are then joined together. This process is called splicing. However, it is not fully understood how the splicing process is coordinated with the other stages of transcription. For example, does splicing occur after the pre-mRNA molecule is completed or while it is still being built? And what controls the order in which these processes occur?
One theory about how the different mRNA-making processes are coordinated is called kinetic competition. This theory states that the fastest process is the most likely to occur, even if the other processes use less energy and so might be expected to be preferred. Alternatively, the different steps may be started and stopped by ‘checkpoints’ that cause the different processes to follow on from each other in a set order.
Coulon et al. used fluorescence microscopy to investigate how mRNA molecules are made during the transcription of a human gene that makes a hemoglobin protein. To make the RNA visible, two different fluorescent markers were introduced into the pre-mRNA that cause different regions of the mRNA to glow in different colors. Coulon et al. made the introns fluoresce red and the exons glow green. Unspliced pre-mRNA molecules contain both introns and exons and so fluoresce in both colors, whereas spliced mRNA molecules contain only exons and so only glow with a green color.
By looking at both the red and green fluorescence signals at the same time, Coulon et al. could see when an intron was spliced out of the pre-mRNA. This revealed that in normal cells, splicing can occur either before or after the RNA is released from where it is transcribed. Thus, splicing and transcription does not follow a set pattern, suggesting that checkpoints do not control the sequence of events. Instead, the fact that a spliced mRNA molecule can be formed in different ways suggests kinetic competition controls the process.
In some cancer cells, there are defects in the cellular machinery that controls splicing. When looking at cells with such a defect, Coulon et al. found that splicing only occurred after transcription was completed. This study thus provides insight into the complex workings of mRNA synthesis and establishes a blueprint for understanding how splicing is impaired in diseases such as cancer.
DOI: http://dx.doi.org/10.7554/eLife.03939.002
doi:10.7554/eLife.03939
PMCID: PMC4210818  PMID: 25271374
transcription; RNA processing; splicing; single-molecule imaging; fluctuation analysis; human
7.  PSF Suppresses Tau Exon 10 Inclusion by Interacting with a Stem-Loop Structure Downstream of Exon 10 
Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA–protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5′ splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.
doi:10.1007/s12031-011-9634-z
PMCID: PMC3893066  PMID: 21881826
Tau; Alternative splicing regulation; Tauopathy; RNA stem-loop secondary structure; Polypyrimidine tract binding protein associated splicing factor (PSF)
8.  The CUGBP2 Splicing Factor Regulates an Ensemble of Branchpoints from Perimeter Binding Sites with Implications for Autoregulation 
PLoS Genetics  2009;5(8):e1000595.
Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter–binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns.
Author Summary
Alternative splicing is a precisely controlled process that determines whether an exon will be included or skipped in the mature mRNA transcript. Factors that control alternative splicing bind to RNA sequence motifs in the exon or flanking introns and guide tissue and developmental specific splicing events. CUGBP2 is a dual functional regulator of alternative splicing that can cause inclusion or skipping of a target exon, depending on the context of its binding motifs. Previously, the mechanisms of regulation by this protein and the positional significance of its target motifs have not been characterized. In this study, the authors dissected the mechanism of exon skipping by CUGBP2 and demonstrate that a specific configuration of motifs at the perimeters of a functional reference point are intimately involved in this event. Furthermore, this mechanism of regulation is shown to have general significance because novel CUGBP2 target exons contain a similar arrangement of motifs. The most interesting of this group is an exon within the CUGBP2 transcript itself. This study underscores the importance of a functional reference point in the specificity of regulation by an alternative splicing factor and reveals a novel autoregulatory role for CUGBP2.
doi:10.1371/journal.pgen.1000595
PMCID: PMC2715136  PMID: 19680430
9.  The Heterogeneous Nuclear Ribonucleoprotein L Is an Essential Component in the Ca2+/Calmodulin-dependent Protein Kinase IV-regulated Alternative Splicing through Cytidine-Adenosine Repeats* 
The Journal of biological chemistry  2008;284(3):1505-1513.
The regulation of gene expression through alternative pre-mRNA splicing is common in metazoans and is often controlled by intracellular signaling pathways that are important in cell physiology. We have shown that the alternative splicing of a number of genes is controlled by membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) through CaMKIV-responsive RNA elements (CaRRE1 and CaRRE2); however, the trans-acting factors remain unknown. Here we show that the heterogeneous nuclear ribonucleoprotein (hnRNP) L is a CaRRE1 binding factor in nuclear extracts. An hnRNP L high affinity CA (cytidine-adenosine) repeat element is sufficient to mediate CaMKIV and hnRNP L repression of splicing in a location (3′-splice site proximity)-dependent way. Depletion of hnRNP L by RNA interference followed by rescue with coexpressed exogenous hnRNP L demonstrates that hnRNP L mediates the CaMKIV-regulated splicing through CA repeats in heterologous contexts. Depletion of hnRNP L also led to increased inclusion of the stress axis-regulated exon and a CA repeat-harboring exon under depolarization or with activated CaMKIV. Moreover, hnRNP L binding to CaRRE1 was increased by CaMKIV and, conversely, was reduced by pretreatments with protein phosphatases. Therefore, hnRNP L is an essential component of CaMKIV-regulated alternative splicing through CA repeats, with its phosphorylation likely playing a critical role.
doi:10.1074/jbc.M805113200
PMCID: PMC3471988  PMID: 19017650 CAMSID: cams2374
10.  Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements 
PLoS Genetics  2007;3(5):e85.
Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.
Author Summary
During RNA splicing, sequences (introns) in a pre-mRNA are excised and discarded, and the remaining sequences (exons) are joined to form the mature RNA. Splicing is regulated not only by the binding of the basic splicing machinery to splice sites located at the exon–intron boundaries, but also by the combined effects of various other splicing factors that bind to a multitude of sequence elements located both in the exons as well as the flanking introns. Instances of alternative splicing, where usage of splice site(s) is incomplete or different between tissues, cell types, or lineages, can be created by the interaction of sequence elements and tissue, cell type, and stage-specific splicing factors. To better understand constitutive and alternative pre-mRNA splicing, the authors describe a comparative genomics approach, using available mammalian genomes, to systematically identify splicing regulatory elements located in the introns proximal to exons. A quarter of the elements were tested experimentally, and most of them altered splicing in human cells. The authors also showed that that the intronic elements are close to tissue-specific alternative exons and are more likely to be located in specific positions in the introns, suggestive of potential regulatory function. These elements are also frequently found in tissue-specific genes, suggesting a coupling between expression and alternative splicing of these genes. Finally, the authors propose a strategy using the elements to identify the binding sites of several splicing factors.
doi:10.1371/journal.pgen.0030085
PMCID: PMC1877881  PMID: 17530930
11.  The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing 
PLoS Genetics  2012;8(7):e1002827.
RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicing-related diseases. We previously isolated a Caenorhabditis elegans mutant defective in an essential gene from a genetic screen for suppressors of the rubberband Unc phenotype of unc-93(e1500) animals. This mutant contains missense mutations in two adjacent codons of the C. elegans microfibrillar-associated protein 1 gene mfap-1. mfap-1(n4564 n5214) suppresses the Unc phenotypes of different rubberband Unc mutants in a pattern similar to that of mutations in the splicing factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). We used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention and exon 3 skipping of tos-1 transcripts in mfap-1(n4564 n5214) animals. Using a yeast two-hybrid screen, we isolated splicing factors as potential MFAP-1 interactors. Our studies indicate that C. elegans mfap-1 encodes a splicing factor that can affect alternative splicing.
Author Summary
RNA splicing removes intervening intronic sequences from pre–mRNA transcripts and joins adjacent exonic sequences to generate functional messenger RNAs. The in vivo functions of numerous factors that regulate splicing remain to be understood. From a genetic screen for suppressors of the rubberband Unc phenotype caused by the Caenorhabditis elegans unc-93(e1500) mutation, we isolated a mutation that affects a highly conserved essential gene, mfap-1. MFAP-1 is a nuclear protein that is broadly expressed. MFAP-1 can affect the alternative splicing of tos-1, an endogenous reporter gene for splicing, and is required for the altered splicing at a cryptic 3′ splice site of tos-1. mfap-1 enhances the effects of the gene uaf-1 (splicing factor U2AF large subunit) in suppressing the rubberband Unc phenotype of unc-93(e1500) animals. Our studies provide in vivo evidence that MFAP-1 functions as a splicing factor.
doi:10.1371/journal.pgen.1002827
PMCID: PMC3400559  PMID: 22829783
12.  The RNA-Binding Protein QKI Suppresses Cancer-Associated Aberrant Splicing 
PLoS Genetics  2014;10(4):e1004289.
Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.
Author Summary
Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicates that splicing program is frequently deregulated during tumorigenesis, and cancer cells favor to produce protein isoforms that can promote growth and survival. Lung cancer is one of the most common cancers and the leading cause of cancer-related death worldwide. Although a number of lung cancer-related splicing events have been detected in several genome-wide analyses, much less is known about how aberrant splicing takes place in lung cancer and how it contributes to tumor development. In this study, we characterized the RNA-binding protein QKI as a new critical regulator of alternative splicing in lung cancer and as a potential marker for prognosis. Genome-wide analysis of QKI-dependent splicing by RNA-Seq identified some cancer-associated splicing changes as its targets. Our results demonstrate that QKI-5 inhibits cancer cell proliferation and prevents inappropriate activation of the Notch signaling pathway by regulating its key target, NUMB. We further showed that QKI-5 represses the inclusion of NUMB alternative exon through competing with a core splicing factor SF1. In summary, our data indicate that down-regulation of QKI causes aberrant splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated repression of Notch signaling.
doi:10.1371/journal.pgen.1004289
PMCID: PMC3983035  PMID: 24722255
13.  NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizure 
eLife  2013;2:e00178.
The neuronal RNA binding protein NOVA regulates splicing, shuttles to the cytoplasm, and co-localizes with target transcripts in dendrites, suggesting links between splicing and local translation. Here we identified >200 transcripts showing NOVA-dependent changes in abundance, but, surprisingly, HITS-CLIP revealed NOVA binds these RNAs in introns rather than 3′ UTRs. This led us to discover NOVA-regulated splicing of cryptic exons within these introns. These exons triggered nonsense mediated decay (NMD), as UPF1 and protein synthesis were required for NOVA's effect on RNA levels. Their regulation was dynamic and physiologically relevant. The NMD exons were regulated by seizures, which also induced changes in Nova subcellular localization and mediated large changes in synaptic proteins, including proteins implicated in familial epilepsy. Moreover, Nova haploinsufficient mice had spontaneous epilepsy. The data reveal a hidden means of dynamic RNA regulation linking electrical activity to splicing and protein output, and of mediating homeostatic excitation/inhibition balance in neurons.
DOI: http://dx.doi.org/10.7554/eLife.00178.001
eLife digest
After the DNA in a gene has been transcribed into messenger RNA, portions of the mRNA called introns are removed, and the remaining stretches of mRNA, which are known as exons, are spliced together. Within eukaryotic cells, a process known as alternative splicing allows a single gene to encode for multiple protein variants by ensuring that some exons are included in the final, modified mRNA, while other exons are excluded. This modified mRNA is then translated into proteins.
Eukaryotic cells also contain proteins that bind to RNA to regulate alternative splicing. These RNA-binding proteins are often found in both the cytoplasm and nucleus of cells, and their involvement in splicing may be linked to other processes in the cell such as mRNA localization and translation. It has also become clear over the past two decades that certain types of RNA-binding proteins, including NOVA proteins, are only found in neurons, and that these proteins have been best characterized as alternative splicing regulators. Recent work has also suggested that they also have important roles in regulating neuronal activity and development, and that their actions in neuronal nuclei and cytoplasm might be coordinated.
Now Eom et al. use the predictive power of a high throughput sequencing and crosslinking method termed HITS-CLIP to show that NOVA proteins can indirectly regulate cytoplasmic mRNA levels by regulating the process of alternative splicing in the nucleus to produce ‘cryptic’ exons in the brains of mice. The presence of these exons in the mRNA leads to the production of premature termination codons in the cytoplasm. These codons trigger a process called nonsense-mediated decay that involves identifying mRNA transcripts that contain nonsense mutations, and then degrading them. These cryptic exons were seen in mice missing the NOVA proteins, where they are expressed in abnormally high levels; in normal mice, these exons have not been seen before, hence they were termed ‘cryptic’.
Eom et al. also show that these cryptic exons are physiologically relevant by inducing epileptic seizures in mice. Following the seizures, they find that the NOVA proteins up-regulate and down-regulate the levels of different cryptic exons, leading to changes in the levels of the proteins encoded by these mRNAs, including proteins that inhibit further seizures. Overall the results indicate that, by controlling the production of various proteins in neurons, these previously unknown cryptic exons have important roles in the workings of the brain.
DOI: http://dx.doi.org/10.7554/eLife.00178.002
doi:10.7554/eLife.00178
PMCID: PMC3552424  PMID: 23359859
HITS-CLIP; Nonsense mediated decay; alternative splicing; RNA regulation; epilepsy; neuronal biology; Mouse
14.  Depolarization-Mediated Regulation of Alternative Splicing 
Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.
doi:10.3389/fnins.2011.00141
PMCID: PMC3246316  PMID: 22207834
alternative splicing; depolarization; RNA-binding proteins; chromatin; histone modification; transcriptional elongation
15.  Alu Exonization Events Reveal Features Required for Precise Recognition of Exons by the Splicing Machinery 
PLoS Computational Biology  2009;5(3):e1000300.
Despite decades of research, the question of how the mRNA splicing machinery precisely identifies short exonic islands within the vast intronic oceans remains to a large extent obscure. In this study, we analyzed Alu exonization events, aiming to understand the requirements for correct selection of exons. Comparison of exonizing Alus to their non-exonizing counterparts is informative because Alus in these two groups have retained high sequence similarity but are perceived differently by the splicing machinery. We identified and characterized numerous features used by the splicing machinery to discriminate between Alu exons and their non-exonizing counterparts. Of these, the most novel is secondary structure: Alu exons in general and their 5′ splice sites (5′ss) in particular are characterized by decreased stability of local secondary structures with respect to their non-exonizing counterparts. We detected numerous further differences between Alu exons and their non-exonizing counterparts, among others in terms of exon–intron architecture and strength of splicing signals, enhancers, and silencers. Support vector machine analysis revealed that these features allow a high level of discrimination (AUC = 0.91) between exonizing and non-exonizing Alus. Moreover, the computationally derived probabilities of exonization significantly correlated with the biological inclusion level of the Alu exons, and the model could also be extended to general datasets of constitutive and alternative exons. This indicates that the features detected and explored in this study provide the basis not only for precise exon selection but also for the fine-tuned regulation thereof, manifested in cases of alternative splicing.
Author Summary
A typical human gene consists of 9 exons around 150 nucleotides in length, separated by introns that are ∼3,000 nucleotides long. The challenge of the splicing machinery is to precisely identify and ligate the exons, while removing the introns. We aimed to understand how the splicing machinery meets this momentous challenge, based on Alu exonization events. Alus are transposable elements, of which approximately one million copies exist in the human genome, a large portion of which within introns. Throughout evolution, some intronic Alus accumulated mutations and became recognized by the splicing machinery as exons, a process termed exonization. Such Alus remain highly similar to their non-exonizing counterparts but are perceived as different by the splicing machinery. By comparing exonizing Alus to their non-exonizing counterparts, we were able to identify numerous features in which they differ and which presumably lead to the recognition only of the former by the splicing machinery. Our findings reveal insights regarding the role of local RNA secondary structures, exon–intron architecture constraints, and splicing regulatory signals. We integrated these features in a computational model, which was able to successfully mimic the function of the splicing machinery and discriminate between true Alu exons and their intronic counterparts, highlighting the functional importance of these features.
doi:10.1371/journal.pcbi.1000300
PMCID: PMC2639721  PMID: 19266014
16.  Genome-Wide Association between Branch Point Properties and Alternative Splicing 
PLoS Computational Biology  2010;6(11):e1001016.
The branch point (BP) is one of the three obligatory signals required for pre-mRNA splicing. In mammals, the degeneracy of the motif combined with the lack of a large set of experimentally verified BPs complicates the task of modeling it in silico, and therefore of predicting the location of natural BPs. Consequently, BPs have been disregarded in a considerable fraction of the genome-wide studies on the regulation of splicing in mammals. We present a new computational approach for mammalian BP prediction. Using sequence conservation and positional bias we obtained a set of motifs with good agreement with U2 snRNA binding stability. Using a Support Vector Machine algorithm, we created a model complemented with polypyrimidine tract features, which considerably improves the prediction accuracy over previously published methods. Applying our algorithm to human introns, we show that BP position is highly dependent on the presence of AG dinucleotides in the 3′ end of introns, with distance to the 3′ splice site and BP strength strongly correlating with alternative splicing. Furthermore, experimental BP mapping for five exons preceded by long AG-dinucleotide exclusion zones revealed that, for a given intron, more than one BP can be chosen throughout the course of splicing. Finally, the comparison between exons of different evolutionary ages and pseudo exons suggests a key role of the BP in the pathway of exon creation in human. Our computational and experimental analyses suggest that BP recognition is more flexible than previously assumed, and it appears highly dependent on the presence of downstream polypyrimidine tracts. The reported association between BP features and the splicing outcome suggests that this, so far disregarded but yet crucial, element buries information that can complement current acceptor site models.
Author Summary
From transcription to translation, the events underlying protein production from DNA sequence are paramount to all aspects of cellular function. Pre-mRNAs in eukaryotes undergo several processing steps prior to their export to the cytoplasm. Among these, splicing – the process of intron removal and exon ligation – has been shown to play a central role in the regulation of gene expression. It has been estimated that more than half of the disease-causing mutations in humans do so by interfering with splicing. The difficulty in describing these disease mechanisms often lies in the low accuracy of the methods for prediction of functional splicing signals in the pre-mRNA. This is especially the case of the branch point, mainly due to its high sequence variability. We have developed a methodology for mammalian branch point prediction based on a machine-learning algorithm, which shows improved accuracy over previous published methods. Moreover, using a combination of experimental and bioinformatics approaches, we uncovered important positional properties of the branch point and shed new light on how some of its features may contribute to the final splicing outcome. These findings might prove useful for a better understanding of how splicing-associated mutations can lead to disease.
doi:10.1371/journal.pcbi.1001016
PMCID: PMC2991248  PMID: 21124863
17.  The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon 
PLoS Genetics  2009;5(11):e1000707.
The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.
Author Summary
This study investigates tissue-specific alternative splicing, which plays a key role in generating diversity in animal cells. We found a new testis-specific exon in a human homologue of the important Drosophila developmental regulator Groucho, which is activated by germ cell RNA binding proteins. By analyzing splicing control of this exon, we elucidated how variations in the activity and expression of splicing regulators together counterbalance splicing activation, and achieve more tightly regulated physiological splicing patterns. We find that although this new human testis-specific exon is not conserved in mice, it is functionally important in that it encodes a peptide which increases the activity of this developmental regulator as a transcriptional repressor. This study provides new insights into how signalling pathways are evolving in human germ cells and the possible molecular defects that might be occurring in infertile men who have genetic deletions of germ cell-specific RNA binding proteins.
doi:10.1371/journal.pgen.1000707
PMCID: PMC2762042  PMID: 19893608
18.  iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions 
PLoS Biology  2010;8(10):e1000530.
Transcriptome-wide analysis of protein-RNA interactions predicts the dual splicing effects of TIA proteins, showing that their local enhancing function is associated with diverse distal splicing silencing effects.
The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5′ splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5′ splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5′ splice sites. Surprisingly, TIA binding at 5′ splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3′ splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5′ splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.
Author Summary
Studies of splicing regulation have generally focused on RNA elements located close to alternative exons. Recently, it has been suggested that splicing of alternative exons can also be regulated by distal regulatory sites, but the underlying mechanism is not clear. The TIA proteins are key splicing regulators that enhance the recognition of 5′ splice sites, and their distal effects have remained unexplored so far. Here, we use a new method to map the positions of TIA-RNA interactions with high resolution on a transcriptome-wide scale. The identified binding positions successfully predict the local enhancing and distal silencing effects of TIA proteins. In particular, we show that TIA proteins can regulate distal alternative 3′ splice sites by binding at the 5′ splice site of the preceding exon. This result suggests that alternative splicing is affected by the timing of alternative exon definition relative to the recognition of the preceding 5′ splice site. These findings highlight the importance of analysing distal regulatory sites in order to fully understand the regulation of alternative splicing.
doi:10.1371/journal.pbio.1000530
PMCID: PMC2964331  PMID: 21048981
19.  A Simple Physical Model Predicts Small Exon Length Variations 
PLoS Genetics  2006;2(4):e45.
One of the most common splice variations are small exon length variations caused by the use of alternative donor or acceptor splice sites that are in very close proximity on the pre-mRNA. Among these, three-nucleotide variations at so-called NAGNAG tandem acceptor sites have recently attracted considerable attention, and it has been suggested that these variations are regulated and serve to fine-tune protein forms by the addition or removal of a single amino acid. In this paper we first show that in-frame exon length variations are generally overrepresented and that this overrepresentation can be quantitatively explained by the effect of nonsense-mediated decay. Our analysis allows us to estimate that about 50% of frame-shifted coding transcripts are targeted by nonsense-mediated decay. Second, we show that a simple physical model that assumes that the splicing machinery stochastically binds to nearby splice sites in proportion to the affinities of the sites correctly predicts the relative abundances of different small length variations at both boundaries. Finally, using the same simple physical model, we show that for NAGNAG sites, the difference in affinities of the neighboring sites for the splicing machinery accurately predicts whether splicing will occur only at the first site, splicing will occur only at the second site, or three-nucleotide splice variants are likely to occur. Our analysis thus suggests that small exon length variations are the result of stochastic binding of the spliceosome at neighboring splice sites. Small exon length variations occur when there are nearby alternative splice sites that have similar affinity for the splicing machinery.
Synopsis
It has recently become clear that splice variation affects most mammalian genes. It is, however, less clear to what extent these splice variations are functional and regulated by the cell as opposed to simply a result of noise in the splicing process.
One of the most frequently observed forms of splice variation are small variations in exon length in which the boundary of an exon is shifted by small amounts between different transcripts. In this work the authors study the statistics of these splice variations in detail, and the results suggest that these variations are mostly the result of noise in the splicing process. In particular, they propose a simple physical model in which the last step of splicing involves the sequence-specific binding of the splicing machinery to the splice site. In this model, small length variations can occur when there are nearby splice sites with comparable affinity for the splicing machinery. The authors show that this model not only accurately predicts the relative abundances of different splice variations but also predicts which splice sites are likely to undergo small exon length variations.
doi:10.1371/journal.pgen.0020045
PMCID: PMC1449888  PMID: 16683028
20.  Cell-to-cell variability of alternative RNA splicing 
The role of mRNA processing in gene expression variability is poorly characterized. This study investigates the extent of cell-to-cell variability of alternative RNA splicing in mammalian cells using single-molecule imaging of CAPRIN1 and MKNK2 splice isoforms.
We applied a single-molecule imaging approach to visualize the alternatively spliced isoforms of two genes, CAPRIN1 and MKNK2, in human cells.We found that cell-to-cell variability in isoform ratios is close to the minimum possible in the absence of feedback in clonal Rpe1 cells, a diploid non-transformed cell line. In contrast, clonal HeLa cells displayed much larger isoform ratio variability between cells.Experimental and theoretical analysis suggests that variability in the regulatory splicing machinery contributes to this difference between cell lines.
Biological gene expression is a complex process which includes transcription, mRNA processing, and translation. As gene expression is a fundamental aspect of biological behavior, a central question within the fields of molecular and cellular biology is how effectively cells control the abundance of their gene expression products, mRNA and protein.
Previous experimental and theoretical studies have shown that there can be substantial cell-to-cell variation in gene expression, even between genetically identical cells grown in uniform conditions. This variation was shown to be important in a variety of biological contexts such as development, virology, immune system function, and cancer treatment. One major source of variability was shown to be transcriptional bursting, or the process in which genes are expressed sporadically separated by long durations of inexpression. Additionally, since the biochemical reactions that govern gene expression are often mediated by molecular species that are present in low numbers, variability can arise from stochastic effects owing to the random chance that an individual biochemical reaction will occur.
The role of mRNA processing in gene expression variability has not been examined thoroughly, particularly with respect to alternative splicing. Alternative RNA splicing is a form of mRNA processing which leads to the synthesis of multiple different mRNAs from a single gene. In this process, the nascent mRNA (pre-mRNA) of a gene contains sequences known as introns that can be excised in different combinations to generate multiple gene products, known as isoforms. As alternative splicing occurs in the vast majority of human genes, it presents a potentially major source of cell-to-cell variability in gene expression.
In this study, we sought to characterize the extent of cell-to-cell variability that arises from alternative RNA splicing. To do so, we utilized a single-molecule imaging approach based on fluorescent in situ hybridization to study the cell-to-cell variability in isoform ratios of two genes, CAPRIN1 and MKNK2, which each contain two splice isoforms (Figure 2 from the manuscript). Using a clonally derived, diploid, non-transformed cell line (Rpe1 cells—retinal pigment epithelial cells), we found that variability is remarkably close to the minimum possible given the probabilistic chance of individual splicing events. In contrast, we found that isoform ratio variability was substantially larger in clonally derived HeLa cells, a cancerous cell line with an unstable karyotype. To explain the differences between the two cell lines, we further examined the potential origins of isoform ratio variability. We first studied several known sources of mRNA variability, such as transcriptional bursting, but found that they did not contribute significantly to the difference between cell lines. However, when we examined the role of splicing factors in controlling cell-to-cell variability, we found that lesser control over the regulation of alternative splicing is likely to be the primary source of this difference.
Cell-to-cell variability in gene expression owing to alternative splicing is an inevitable feature of biology. Since spliced isoforms can have different and even opposing cellular functions, it would be interesting to see if such variability can have phenotypic consequences in various biological settings. We anticipate that future work will shed light on the extent of cell-to-cell variability of alternative splicing for additional genes, and may identify splicing events where heterogeneity has an important functional role.
Heterogeneity in the expression levels of mammalian genes is large even in clonal populations and has phenotypic consequences. Alternative splicing is a fundamental aspect of gene expression, yet its contribution to heterogeneity is unknown. Here, we use single-molecule imaging to characterize the cell-to-cell variability in mRNA isoform ratios for two endogenous genes, CAPRIN1 and MKNK2. We show that isoform variability in non-transformed, diploid cells is remarkably close to the minimum possible given the stochastic nature of individual splicing events, while variability in HeLa cells is considerably higher. Analysis of the potential sources of isoform ratio heterogeneity indicates that a difference in the control over splicing factor activity is one origin of this increase. Our imaging approach also visualizes non-alternatively spliced mRNA and active transcription sites, and yields spatial information regarding the relationship between splicing and transcription. Together, our work demonstrates that mammalian cells minimize fluctuations in mRNA isoform ratios by tightly regulating the splicing machinery.
doi:10.1038/msb.2011.32
PMCID: PMC3159976  PMID: 21734645
alternative splicing; cell-to-cell variability; co-transcriptional splicing; gene expression
21.  Evolution of Nova-Dependent Splicing Regulation in the Brain 
PLoS Genetics  2007;3(10):e173.
A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.
Author Summary
Alternative splicing generates different mRNA isoforms from a single gene and thus increases the number of proteins a cell can produce. This is particularly important in the brain, which possesses a number of brain-specific splicing factors. In this study, we have looked at evolution of brain-specific splicing regulation by one such factor, Nova. Previous studies have identified ∼100 alternative exons that are regulated by Nova in mouse brain. We find that the Nova protein sequence changed little during vertebrate evolution from fish to human, whereas the RNA targets themselves have evolved significantly. Interestingly, the presence of conserved Nova binding elements in an RNA transcript in most cases correlates with conservation of brain-specific splicing. In addition, the evolution of Nova-dependent splicing relates to the functions encoded by the target RNAs, such that Nova-regulated splicing of RNAs encoding core roles such as synaptic adhesion, ion channel, and cytoskeletal proteins is on average more conserved than splicing of the RNAs encoding regulatory roles, such as transmembrane receptor and signal transduction proteins.
doi:10.1371/journal.pgen.0030173
PMCID: PMC2014790  PMID: 17937501
22.  A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression 
PLoS Genetics  2013;9(10):e1003856.
The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function.
Author Summary
The accurate removal of intervening sequences (introns) from precursor messenger RNAs (pre-mRNAs) represents an essential step in the expression of most eukaryotic protein-coding genes. Alternative splicing can create from a single primary transcript various mature mRNAs with diverse, sometimes even antagonistic, biological functions. Many human diseases are based on alternative-splicing defects, and most interestingly, certain defects are caused by mutations in general splicing factors that participate in each splicing event. To address the question of how a general splicing factor can regulate alternative splicing events, here we investigated the regulatory role of the U1C protein, a specific component of the U1 small nuclear ribonucleoprotein (snRNP) and important in initial 5′ splice site recognition. Our RNA-Seq analysis demonstrated that U1C affects more than 300 cases of alternative splicing in the human system. One U1C target, U1-70K, appeared to be particularly interesting, because both protein products are components of the U1 snRNP and functionally depend on each other. Analyzing the mechanistic basis of this intra-U1 snRNP cross-regulation, we discovered a U1C-dependent alternative splicing switch in the U1-70K pre-mRNA that regulates U1-70K expression. In sum, this feedback loop controls and links U1C and U1-70K homeostasis to guarantee correct U1 snRNP assembly and function.
doi:10.1371/journal.pgen.1003856
PMCID: PMC3798272  PMID: 24146627
23.  Developmental Control of CaV1.2 L-Type Calcium Channel Splicing by Fox Proteins▿ †  
Molecular and Cellular Biology  2009;29(17):4757-4765.
CaV1.2 voltage-gated calcium channels play critical roles in the control of membrane excitability, gene expression, and muscle contraction. These channels show diverse functional properties generated by alternative splicing at multiple sites within the CaV1.2 pre-mRNA. The molecular mechanisms controlling this splicing are not understood. We find that two exons in the CaV1.2 channel are controlled in part by members of the Fox family of splicing regulators. Exons 9* and 33 confer distinct electrophysiological properties on the channel and show opposite patterns of regulation during cortical development, with exon 9* progressively decreasing its inclusion in the CaV1.2 mRNA over time and exon 33 progressively increasing. Both exons contain Fox protein binding elements within their adjacent introns, and Fox protein expression is induced in cortical neurons in parallel with the changes in CaV1.2 splicing. We show that knocking down expression of Fox proteins in tissue culture cells has opposite effects on exons 9* and 33. The loss of Fox protein increases exon 9* splicing and decreases exon 33, as predicted by the positions of the Fox binding elements and by the pattern of splicing in development. Conversely, overexpression of Fox1 and Fox2 proteins represses exon 9* and enhances exon 33 splicing in the endogenous CaV1.2 mRNA. These effects of Fox proteins on exons 9* and 33 can be recapitulated in transfected minigene reporters. Both the repressive and the enhancing effects of Fox proteins are dependent on the Fox binding elements within and adjacent to the target exons, indicating that the Fox proteins are directly regulating both exons. These results demonstrate that the Fox protein family is playing a key role in tuning the properties of CaV1.2 calcium channels during neuronal development.
doi:10.1128/MCB.00608-09
PMCID: PMC2725710  PMID: 19564422
24.  ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation 
PLoS Genetics  2013;9(10):e1003895.
The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. Here we report a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, we identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. We demonstrated that FUS is a repressor of exon 7 splicing and that the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, we examined whether cells expressing these mutants would be deficient in repressing exon 7 splicing. We showed that FUS harbouring R521G, R522G or ΔExon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) directly correlated with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. We showed that exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.
Author Summary
FUS/TLS is a frequently mutated gene in amyotrophic lateral sclerosis (ALS). ALS, also known as Lou Gehrig's disease, is characterized by a progressive degeneration of motor neurons. The abnormal cytoplasmic accumulation of mutant FUS protein is a characteristic pathology of ALS; however, recent evidence increasingly suggests deficiencies in FUS nuclear function may also contribute to neurodegeneration in ALS. Here we report a novel autoregulatory mechanism of FUS by alternative splicing and nonsense mediated decay (NMD). We show FUS binds to exon 7 and flanking introns of its own pre-mRNAs. This results in exon skipping, inducing a reading frame shift and subsequent degradation of the splice variants. As such, this mechanism provides a feedback loop that controls the homeostasis of FUS protein levels. This balance is disrupted in ALS-associated FUS mutants, which are deficient in nuclear localization and FUS-dependent alternative splicing. As a result, the abnormal accumulation of mutant FUS protein in ALS neurons goes unchecked and uncontrolled. Our study provides novel insight into the molecular mechanism by which FUS regulates gene expression and new understanding of the role of FUS in disease at the molecular level. This may lead to new potential therapeutic targets for the treatment of ALS.
doi:10.1371/journal.pgen.1003895
PMCID: PMC3814325  PMID: 24204307
25.  Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA. 
Nucleic Acids Research  1988;16(17):8443-8465.
The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.
Images
PMCID: PMC338569  PMID: 3419923

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