Figure 3 - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
![Expression of cTAF1, TAF1-34ʹ and SRRM4 mRNAs during in vitro neuronal differentiation. Mouse embryonic stem cells (mESCs) were differentiated towards cortical glutamatergic neurons as depicted in panel A (adapted from [15]). The percentage-splicedin (PSI) of microexon 34ʹ (B) and Srrm4 expression level (C) were calculated at different time points (mESCs: day −8; neuroepithelial stem cells -NESCs: day −4; radial glia -RG: day 0; neuronal differentiation stage I-II: day 1; stage III-IV: day 7; stage IV-V: days 16, 21, 28). Dotted lines in panels B and C indicate the data trend line. cRPKM indicates corrected reads per kilo base per million mapped reads. Microexon 34ʹ incorporation in TAF1 mRNAs (D) correlates with SRRM4 expression (E) in the LUHMES differentiation assay.](publication/336106479/figure/fig3/AS:807794190057480@1569604551859/Expression-of-cTAF1-TAF1-34-and-SRRM4-mRNAs-during-in-vitro-neuronal-differentiation.png)
Expression of cTAF1, TAF1-34ʹ and SRRM4 mRNAs during in vitro neuronal differentiation. Mouse embryonic stem cells (mESCs) were differentiated towards cortical glutamatergic neurons as depicted in panel A (adapted from [15]). The percentage-splicedin (PSI) of microexon 34ʹ (B) and Srrm4 expression level (C) were calculated at different time points (mESCs: day −8; neuroepithelial stem cells -NESCs: day −4; radial glia -RG: day 0; neuronal differentiation stage I-II: day 1; stage III-IV: day 7; stage IV-V: days 16, 21, 28). Dotted lines in panels B and C indicate the data trend line. cRPKM indicates corrected reads per kilo base per million mapped reads. Microexon 34ʹ incorporation in TAF1 mRNAs (D) correlates with SRRM4 expression (E) in the LUHMES differentiation assay.
Source publication
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 34ʹ is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this...
Contexts in source publication
Context 1
... brain suggested that these two isoforms display a temporal regulation. To investigate when during neural development Taf1-34ʹ first emerges, we examined the expression profile of this isoform using RNA-seq data collected at different time points during differentiation of mouse embryonic stem cells (mESCs) into cortical glutamatergic neurons [14] (Fig. 3A). Inclusion of microexon 34ʹ was evaluated using the Percentage-Spliced-In (PSI) metric, which determines the percentage of Taf1 mRNAs containing microexon 34ʹ. This study indicated that microexon 34ʹ is incorporated into Taf1 mRNA at the beginning of neuronal differentiation, reaching the highest value in mature post-mitotic neurons ( ...
Context 2
... (Fig. 3A). Inclusion of microexon 34ʹ was evaluated using the Percentage-Spliced-In (PSI) metric, which determines the percentage of Taf1 mRNAs containing microexon 34ʹ. This study indicated that microexon 34ʹ is incorporated into Taf1 mRNA at the beginning of neuronal differentiation, reaching the highest value in mature post-mitotic neurons ( Fig. 3B). At earlier stages, when neuroepithelial cells and radial glia were undergoing divisions, microexon 34ʹ was not detectable. The expression of Srrm4 mRNA began in glial daughters and peaked in the early phase of neuronal maturation. As neuronal differentiation proceeded, Srrm4 expression decreased (Fig. 3C). These results indicate a ...
Context 3
... value in mature post-mitotic neurons ( Fig. 3B). At earlier stages, when neuroepithelial cells and radial glia were undergoing divisions, microexon 34ʹ was not detectable. The expression of Srrm4 mRNA began in glial daughters and peaked in the early phase of neuronal maturation. As neuronal differentiation proceeded, Srrm4 expression decreased (Fig. 3C). These results indicate a switch from cTaf1 to Taf1-34ʹ expression in the early phase of neuronal differentiation with a further increase in Taf1-34ʹ expression in mature neurons. Interestingly, Srrm4 expression peaks with the earliest detection of microexon 34ʹ, suggesting that Srrm4 initiates the transition of cTaf1 to Taf1-34ʹ mRNAs ...
Context 4
... in a tetracycline-dependent manner, which induces LUHMES cells to exit cell cycle and differentiate into morphologically and biochemically post-mitotic dopamine-like neurons [15,16]. Immunoblot analysis showed that endogenous SRRM4 was detectable at day 2 of differentiation and its expression was decreasing in later differentiation stages (Fig. 3E). Consistent with SRRM4 expression, the incorporation of microexon 34ʹ into TAF1 mRNA was observed only in the latest differentiation points (day 4 and day 6) (Fig. ...
Context 5
... neurons [15,16]. Immunoblot analysis showed that endogenous SRRM4 was detectable at day 2 of differentiation and its expression was decreasing in later differentiation stages (Fig. 3E). Consistent with SRRM4 expression, the incorporation of microexon 34ʹ into TAF1 mRNA was observed only in the latest differentiation points (day 4 and day 6) (Fig. ...
Context 6
... 34ʹ inclusion lagged only shortly behind GFP-SRRM4 induction, which indicates a rapid turnover of the pool of TAF1 mRNAs (Fig. 4F). We examined two other microexon splicing events, the incorporation of microexon 8A in KDM1A and microexon 16 in DAAM1 attributed to SRRM4 and observed inclusion of both microexons into their respective mRNAs upon SRMM4 induction in HeLa cells ( Supplementary Fig. S3C and S3D). ...
Context 7
... indicates corrected reads per kilo base per million mapped reads. hierarchy of SRRM4 interactors by iBAQ-based quantitative mass spectrometry (qMS) of GFP-SRRM4 purified from nuclear extracts of DOX-induced HeLa cells ( Supplementary Fig. S3E). This procedure identified U2 snRNP auxiliary splicing factor, composed of the U2AF1 (U2AF35)/U2AF2 (U2AF65), as a major interactor of SRRM4. ...
Context 8
... alternative expression of TAF1 and TAF1-34ʹ isoforms is restricted to specific cell types in the brain and we show that the neuronal splicing factor SRRM4 directs this switch. Using both ISH and IHC, we could confirm the overlapping expression pattern between Srrm4 and Taf1-34ʹ. Interestingly, while the cortex showed very high correlation (Fig. 3B and 3C), the striatum sample challenged this finding, showing strong immunoreactivity for Taf1-34ʹ but a weak Srrm4 staining (Fig. 2B''-C''). This data could indicate that a low expression of Srrm4, not detectable by IHC, is sufficient to promote Taf1-34ʹ microexon inclusion. On the other hand, this could also suggest that an intricate ...
Citations
... The misregulation of microexons is involved in abnormal brain development, autism, and various cancers. [9][10][11][12][13][14][15] Thus far, the regulatory mechanisms on microexon splicing have been studied with multiple of 3 bp (3x bp) microexons. RBPs, which regulate alternative splicing, are involved in microexon splicing in tissue and disease-specific manners. ...
... 9 SRRM4, a brain-specific RBP, stimulates the inclusion of microexon in the brain and various cancers. 10,12,15,16 QUAKING (QKI) plays important roles in microglia homeostasis by regulating alternative splicing of Rho GTPase pathway-related microexons. 14 Ubiquitously present RBPs, Srsf11, and Rnps1 identified by genome-wide CRISPR-Cas9, preferentially regulate neuronal microexons. ...
Alternative splicing of microexons (3-30 base pairs [bp]) is involved in important biological processes in brain development and human cancers. However, understanding a splicing process of non-3x bp microexons is scarce. We showed that 4 bp microexon of mitochondrial pyruvate carrier1 (MPC1) is constitutively included in mRNA. Based on our studies with minigene and exon island constructs, we found the strong exon definition region in the proximal introns bordering MPC1 microexon. Ultimately, we defined a nucleotide fragment from the 3'ss 67 bp of MPC1 microexon to the 5'ss consensus sequence, as a core exon island, which can concatenate its microexon and neighboring exons by splicing. Furthermore, we showed that insertion of the core exon island into a target exon or intron induced skip the target exon or enhance the splicing of an adjacent exon, respectively. Collectively, we suggest that the exon island derived from MPC1 microexon modifies genuine splicing patterns depending on its position, thereby providing insights on strategies for splicing-mediated gene correction.
... In mice and human several ubiquitously expressed TAF1 isoforms have been described, while neuronal tissue expresses an isoform that includes a 6-nucleotide long microexon (Makino et al., 2007). Microexon inclusion is temporally regulated and the resulting neuronal isoform N-TAF1 is predominantly expressed in postmitotic neurons (Capponi et al., 2020). It was postulated by the authors that such cell-type specific splicing events could contribute to tissue-specific disease phenotypes of ubiquitously expressed genes. ...
Transcription pause-release is an important, highly regulated step in the control of gene expression. Modulated by various factors, it enables signal integration and fine-tuning of transcriptional responses. Mutations in regulators of pause-release have been identified in a range of neurodevelopmental disorders that have several common features affecting multiple organ systems. This review summarizes current knowledge on this novel subclass of disorders, including an overview of clinical features, mechanistic details, and insight into the relevant neurodevelopmental processes.
... Other studies have shown the functional role of neuronal microexons in chromatin regulation and transcription [133], axon growth and synapse formation [78,124,134,135], neuronal differentiation [136], microglia homeostasis [122] and animal behaviour [77,133,137]. In conclusion, splicing of neuronal microexons represents an evolutionarily conserved mechanism of gene expression regulation which contributes to the functional complexity of the CNS. ...
The advance of experimental and computational techniques has allowed us to highlight the existence of numerous different mechanisms of RNA maturation, which have been so far unknown. Besides canonical splicing, consisting of the removal of introns from pre-mRNA molecules, non-canonical splicing events may occur to further increase the regulatory and coding potential of the human genome. Among these, splicing of microexons, recursive splicing and biogenesis of circular and chimeric RNAs through back-splicing and trans-splicing processes, respectively, all contribute to expanding the repertoire of RNA transcripts with newly acquired regulatory functions. Interestingly, these non-canonical splicing events seem to occur more frequently in the central nervous system, affecting neuronal development and differentiation programs with important implications on brain physiology. Coherently, dysregulation of non-canonical RNA processing events is associated with brain disorders, including brain tumours. Herein, we summarize the current knowledge on molecular and regulatory mechanisms underlying canonical and non-canonical splicing events with particular emphasis on cis-acting elements and trans-acting factors that all together orchestrate splicing catalysis reactions and decisions. Lastly, we review the impact of non-canonical splicing on brain physiology and pathology and how unconventional splicing mechanisms may be targeted or exploited for novel therapeutic strategies in cancer.
... The splicing of microexons was studied in animals, especially in neurogenesis 10,21 . The inclusion of microexon depends on the level of cell specificity and development and is mediated by cisregulatory elements, such as exonic splicing enhancers (ESEs) and intronic splicing enhancers (ISEs) 22 . ...
... The splicing of microexons has been studied in neurogenesis for animals 10,21 . In animals, the sizes of microexons are usually multiples of 3-nt and in-frame 9,10 . ...
It is challenging to identify the smallest microexons (≤15-nt) due to their small size. Consequently, these microexons are often misannotated or missed entirely during genome annotation. Here, we develop a pipeline to accurately identify 2,398 small microexons in 10 diverse plant species using 990 RNA-seq datasets, and most of them have not been annotated in the reference genomes. Analysis reveals that microexons tend to have increased detained flanking introns that require post-transcriptional splicing after polyadenylation. Examination of 45 conserved microexon clusters demonstrates that microexons and associated gene structures can be traced back to the origin of land plants. Based on these clusters, we develop an algorithm to genome-wide model coding microexons in 132 plants and find that microexons provide a strong phylogenetic signal for plant organismal relationships. Microexon modeling reveals diverse evolutionary trajectories, involving microexon gain and loss and alternative splicing. Our work provides a comprehensive view of microexons in plants.
... 17 The neuron-specific splicing factor Serine/Arginine Repetitive Matrix 4 (SRRM4) is the main driver of brain-specific microexon inclusion in general 18 and of microexon 34 0 incorporation into TAF1 mRNAs in particular. 19 The current paradigm for the pathomechanism of XDP is a downregulation of TAF1 isoforms containing microexon 34 0 , which would be due to the XDP-specific SVA insertion within intron 32. 8 This paradigm depends on TAF1 mRNA analysis of one XDP brain versus one control, showing 40-folds reduction in the striatum (ST) and about 5-fold reduction in the cortex. 8 Similar observations were reported for XDP iPSCs-derived neural stem cells (NSCs). ...
... The pcDNA5/FRT/TO/GFP-SRRM4 expression vector has been described. 19 Human embryonic kidney cells 293T, mouse neuroblastoma cells N2a and human cervical cancer cells HeLa were cultured in DMEM containing 4.5 g/l of glucose (Lonza), supplemented with 10% (v/v) fetal bovine serum (Lonza). Transient transfection was performed using FuGENE HD Transfection Reagent (Promega). ...
... Doxycyclin (DOX)-inducible HeLa derivatives expressing GFP-SRRM4 were previously described. 19 DOX-inducible N2a were a kind gift from Dr Blencowe (University of Toronto, Canada) and N2a derivatives were generated and induced as described. 19 by SDS-PAGE followed by immunoblotting. ...
X-linked Dystonia-Parkinsonism is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In X-linked Dystonia-Parkinsonism the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34'. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in X-linked Dystonia-Parkinsonism and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34’ was further investigated in cellular assays. Subsequently, microexon 34’ incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from X-linked Dystonia-Parkinsonism and control brains tissues. Using cell-based splicing assays we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34’. In addition, we show that (1) microexon 34’-containing TAF1 mRNAs are detected at similar levels in X-linked Dystonia-Parkinsonism as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between X-linked Dystonia-Parkinsonism and controls brains. These results indicate that microexon 34’ incorporation into TAF1 mRNA is not affected in X-linked Dystonia-Parkinsonism brains. Our findings shift the current paradigm of X-linked Dystonia-Parkinsonism by discounting alternative splicing of TAF1 microexon 34’ as the molecular basis for this disease.
... Furthermore, they are enriched for lengths that are multiple of 3 nucleotides and are thus likely to produce alternative protein isoforms [29]. Microexons impact on specific protein regulatory domains, are associated with late neurogenesis and appear altered in neurological disorders [29][30][31][32][33] (Figure 1B). Some splicing errors can cause frameshift and premature protein truncation, thus resulting in transcripts that are recognized by the cellular mRNA control machinery and are degraded by nonsense-mediated decay (NMD). ...
Alternative splicing of mRNA is an essential mechanism to regulate and increase the diversity of the transcriptome and proteome. Alternative splicing frequently occurs in a tissue- or time-specific manner, contributing to differential gene expression between cell types during development. Neural tissues present extremely complex splicing programs and display the highest number of alternative splicing events. As an extension of the central nervous system, the retina constitutes an excellent system to illustrate the high diversity of neural transcripts. The retina expresses retinal specific splicing factors and produces a large number of alternative transcripts, including exclusive tissue-specific exons, which require an exquisite regulation. In fact, a current challenge in the genetic diagnosis of inherited retinal diseases stems from the lack of information regarding alternative splicing of retinal genes, as a considerable percentage of mutations alter splicing or the relative production of alternative transcripts. Modulation of alternative splicing in the retina is also instrumental in the design of novel therapeutic approaches for retinal dystrophies, since it enables precision medicine for specific mutations.
Background:
X-linked dystonia parkinsonism is a generalized, progressive dystonia followed by parkinsonism with onset in adulthood and accompanied by striatal neurodegeneration. Causative mutations are located in a noncoding region of the TATA-box binding protein-associated factor 1 (TAF1) gene and result in aberrant splicing. There are 2 major TAF1 isoforms that may be decreased in symptomatic patients, including the ubiquitously expressed canonical cTAF1 and the neuronal-specific nTAF1.
Objective:
The objective of this study was to determine the behavioral and transcriptomic effects of decreased cTAF1 and/or nTAF1 in vivo.
Methods:
We generated adeno-associated viral (AAV) vectors encoding microRNAs targeting Taf1 in a splice-isoform selective manner. We performed intracerebroventricular viral injections in newborn mice and rats and intrastriatal infusions in 3-week-old rats. The effects of Taf1 knockdown were assayed at 4 months of age with evaluation of motor function, histology, and RNA sequencing of the striatum, followed by its validation.
Results:
We report motor deficits in all cohorts, more pronounced in animals injected at P0, in which we also identified transcriptomic alterations in multiple neuronal pathways, including the cholinergic synapse. In both species, we show a reduced number of striatal cholinergic interneurons and their marker mRNAs after Taf1 knockdown in the newborn.
Conclusion:
This study provides novel information regarding the requirement for TAF1 in the postnatal maintenance of striatal cholinergic neurons, the dysfunction of which is involved in other inherited forms of dystonia. © 2021 International Parkinson and Movement Disorder Society.
The discovery and characterization of a network of highly conserved neuronal microexons have provided fundamental new insight into mechanisms underlying nervous system development and function, as well as an important basis for pathway convergence in autism spectrum disorder. In the past few years, considerable progress has been made in comprehensively determining the repertoires of factors that control neuronal microexons. These results have illuminated molecular mechanisms that activate the splicing of microexons, including those that control gene expression programs critical for neurogenesis, as well as synaptic protein translation and neuronal activity. Remarkably, individual disruption of specific microexons in these pathways results in autism-like phenotypes and cognitive impairment in mice. This review discusses these findings and their implications for delivering new therapeutic strategies for neurological disorders.
