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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...
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... in neuron-rich regions relative to the low levels in the glial-rich corpus callosum ( Fig. 1C and 1C'). Srrm4-positive cells were also present along the ventricle wall and SVZ (Fig. 1C''). The mRNA ISH results were validated by immunohistochemical analyses (IHC) of different mouse brain regions by cTaf1-, Taf1-34ʹ-and Srrm4-specific antibodies (Fig. 2). The affinity-purified Taf1 antibodies developed in this study were directed against the region spanning microexon 34ʹ as the isoform-specific epitope, which is identical between mouse and human TAF1-34ʹ. The sera displayed high specificity in the detection by IHC and immunoblotting of both endogenous ( Fig. 2A-C'') and ectopically ...
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... Srrm4-specific antibodies (Fig. 2). The affinity-purified Taf1 antibodies developed in this study were directed against the region spanning microexon 34ʹ as the isoform-specific epitope, which is identical between mouse and human TAF1-34ʹ. The sera displayed high specificity in the detection by IHC and immunoblotting of both endogenous ( Fig. 2A-C'') and ectopically expressed proteins ( Fig. 2D-H and Supplementary Fig. 1), with very limited, if any cross-reactivity when tested against the counterpart isoform ( Fig. 2D-H and Supplementary Fig. 1). As additional validation, we performed IHC and confirmed that the antibodies against Taf1-34ʹ and Srrm4 were selectively immunoreactive ...
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... epitope, which is identical between mouse and human TAF1-34ʹ. The sera displayed high specificity in the detection by IHC and immunoblotting of both endogenous ( Fig. 2A-C'') and ectopically expressed proteins ( Fig. 2D-H and Supplementary Fig. 1), with very limited, if any cross-reactivity when tested against the counterpart isoform ( Fig. 2D-H and Supplementary Fig. 1). As additional validation, we performed IHC and confirmed that the antibodies against Taf1-34ʹ and Srrm4 were selectively immunoreactive with cell nuclei in neuron-rich regions of the brain, whereas the antibody against cTaf1 was ubiquitously immunoreactive across neuronal and non-neuronal cell nuclei ...
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... We next examined the distribution of cTaf1-, Taf1-34ʹ-and Srrm4 immunoreactivity within the brain. Consistent with the mRNA results, cTaf1 protein expression was detected in the nuclei of many cells throughout the brain, including in cells of the glial-rich corpus callosum and in post-mitotic neurons within the cerebral cortex and the striatum ( Fig. 2A-A''). By contrast, Taf1-34ʹ expression was selectively enriched in neuronal nuclei ( Fig. 2B and B'') and was low within the corpus callosum (Fig. 2B'). The expression of Srrm4 also appeared enriched in neuronal nuclei and at low levels in the corpus callosum ( Fig. 2C-2C''). Magnification of sections at levels including the striatum ...
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... brain. Consistent with the mRNA results, cTaf1 protein expression was detected in the nuclei of many cells throughout the brain, including in cells of the glial-rich corpus callosum and in post-mitotic neurons within the cerebral cortex and the striatum ( Fig. 2A-A''). By contrast, Taf1-34ʹ expression was selectively enriched in neuronal nuclei ( Fig. 2B and B'') and was low within the corpus callosum (Fig. 2B'). The expression of Srrm4 also appeared enriched in neuronal nuclei and at low levels in the corpus callosum ( Fig. 2C-2C''). Magnification of sections at levels including the striatum showed that expression was high in the neocortex (Fig. 2C) but sparsely distributed in ...
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... was detected in the nuclei of many cells throughout the brain, including in cells of the glial-rich corpus callosum and in post-mitotic neurons within the cerebral cortex and the striatum ( Fig. 2A-A''). By contrast, Taf1-34ʹ expression was selectively enriched in neuronal nuclei ( Fig. 2B and B'') and was low within the corpus callosum (Fig. 2B'). The expression of Srrm4 also appeared enriched in neuronal nuclei and at low levels in the corpus callosum ( Fig. 2C-2C''). Magnification of sections at levels including the striatum showed that expression was high in the neocortex (Fig. 2C) but sparsely distributed in striatal cell nuclei ( Fig. 2C''). The ISH and IHC performed on ...
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... and in post-mitotic neurons within the cerebral cortex and the striatum ( Fig. 2A-A''). By contrast, Taf1-34ʹ expression was selectively enriched in neuronal nuclei ( Fig. 2B and B'') and was low within the corpus callosum (Fig. 2B'). The expression of Srrm4 also appeared enriched in neuronal nuclei and at low levels in the corpus callosum ( Fig. 2C-2C''). Magnification of sections at levels including the striatum showed that expression was high in the neocortex (Fig. 2C) but sparsely distributed in striatal cell nuclei ( Fig. 2C''). The ISH and IHC performed on mouse brains indicated that cTaf1 and Taf1-34ʹ have different expression patterns within the forebrain. We therefore ...
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... was selectively enriched in neuronal nuclei ( Fig. 2B and B'') and was low within the corpus callosum (Fig. 2B'). The expression of Srrm4 also appeared enriched in neuronal nuclei and at low levels in the corpus callosum ( Fig. 2C-2C''). Magnification of sections at levels including the striatum showed that expression was high in the neocortex (Fig. 2C) but sparsely distributed in striatal cell nuclei ( Fig. 2C''). The ISH and IHC performed on mouse brains indicated that cTaf1 and Taf1-34ʹ have different expression patterns within the forebrain. We therefore validated this differential distribution in the human brain by analysing RNA-seq data ( Supplementary Fig. S2, panels A-B) and ...
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... B'') and was low within the corpus callosum (Fig. 2B'). The expression of Srrm4 also appeared enriched in neuronal nuclei and at low levels in the corpus callosum ( Fig. 2C-2C''). Magnification of sections at levels including the striatum showed that expression was high in the neocortex (Fig. 2C) but sparsely distributed in striatal cell nuclei ( Fig. 2C''). The ISH and IHC performed on mouse brains indicated that cTaf1 and Taf1-34ʹ have different expression patterns within the forebrain. We therefore validated this differential distribution in the human brain by analysing RNA-seq data ( Supplementary Fig. S2, panels A-B) and RT-PCR ( Supplementary Fig. S2, panels C-D) from specimens ...
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... ISH and IHC performed on mouse brains indicated that cTaf1 and Taf1-34ʹ have different expression patterns within the forebrain. We therefore validated this differential distribution in the human brain by analysing RNA-seq data ( Supplementary Fig. S2, panels A-B) and RT-PCR ( Supplementary Fig. S2, panels C-D) from specimens from the striatum (caudate nucleus and putamen), neocortex, motor cortex, and thalamus. Consistent with the ISH data from the mouse brain, we confirmed that the inclusion of microexon 34ʹ is more prominent in the neocortex than within the striatum, while not detectable in the thalamus. ...
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... ISH and IHC performed on mouse brains indicated that cTaf1 and Taf1-34ʹ have different expression patterns within the forebrain. We therefore validated this differential distribution in the human brain by analysing RNA-seq data ( Supplementary Fig. S2, panels A-B) and RT-PCR ( Supplementary Fig. S2, panels C-D) from specimens from the striatum (caudate nucleus and putamen), neocortex, motor cortex, and thalamus. Consistent with the ISH data from the mouse brain, we confirmed that the inclusion of microexon 34ʹ is more prominent in the neocortex than within the striatum, while not detectable in the thalamus. ...
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... 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 network of AS regulation initiated by Srrm4 is sustained by other splicing factors that have functions overlapping with Srrm4. This second ...
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.
