Neuronal cell-type-specific alternative splicing: A mechanism for specifying connections in the brain?

The enormous cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell-type–specific gene-regulatory programs at the molecular level. Although prevalent in the brain, the contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here, we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single-cell RNA-sequencing data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class. These programs consist of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2, and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs contribute to splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which may provide a molecular mechanism to specify neuronal identity and function that are orthogonal to established classifications based on transcriptional regulation.

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Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

10.1101/2021.08.03.454921 ◽

2021 ◽

Author(s):

Sruti Rayaprolu ◽

Sara Bitarafan ◽

Ranjita Betarbet ◽

Sydney N Sunna ◽

Lihong Cheng ◽

...

Keyword(s):

Mouse Brain ◽

Neurological Diseases ◽

Neuronal Cell ◽

Cell Types ◽

Proteomic Profiling ◽

Cell Type ◽

Specific Expression ◽

Cell Type Specific ◽

The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

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Long Non-Coding RNA Expression Levels Modulate Cell-Type-Specific Splicing Patterns by Altering Their Interaction Landscape with RNA-Binding Proteins

Genes ◽

10.3390/genes10080593 ◽

2019 ◽

Vol 10 (8) ◽

pp. 593 ◽

Cited By ~ 4

Author(s):

Felipe Wendt Porto ◽

Swapna Vidhur Daulatabad ◽

Sarath Chandra Janga

Keyword(s):

Alternative Splicing ◽

Cell Lines ◽

Rna Binding ◽

Cell Type ◽

Specific Manner ◽

Alternatively Spliced ◽

A Cell ◽

Specific Alternative ◽

Cell Type Specific ◽

Rna Interaction

Recent developments in our understanding of the interactions between long non-coding RNAs (lncRNAs) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein–RNA interaction networks. Analysis of alternative splicing events across 39 lncRNA knockdown and wildtype RNA-sequencing datasets from three human cell lines—HeLa (cervical cancer), K562 (myeloid leukemia), and U87 (glioblastoma)—resulted in the high-confidence (false discovery rate (fdr) < 0.01) identification of 11,630 skipped exon events and 5895 retained intron events, implicating 759 genes to be impacted at the post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockdown were specific to the cell type. In tandem, the functions annotated to the genes affected by alternative splicing across each lncRNA knockdown also displayed cell-type specificity. To understand the mechanism behind this cell-type-specific alternative splicing pattern, we analyzed RNA-binding protein (RBP)–RNA interaction profiles across the spliced regions in order to observe cell-type-specific alternative splice event RBP binding preference. Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron–exon junctions in a cell-type-specific manner. The cellular functions affected by alternative splicing were also affected in a cell-type-specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs and their downstream functions. We propose that such lncRNA sponges can extensively rewire post-transcriptional gene regulatory networks by altering the protein–RNA interaction landscape in a cell-type-specific manner.

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Alternative Splicing Coupled Nonsense-Mediated Decay Generates Neuronal Cell Type-Specific Expression of SLM Proteins

Journal of Neuroscience ◽

10.1523/jneurosci.3395-14.2014 ◽

2014 ◽

Vol 34 (50) ◽

pp. 16755-16761 ◽

Cited By ~ 25

Author(s):

L. Traunmuller ◽

C. Bornmann ◽

P. Scheiffele

Keyword(s):

Alternative Splicing ◽

Neuronal Cell ◽

Cell Type ◽

Specific Expression ◽

Nonsense Mediated Decay ◽

Cell Type Specific Expression ◽

Cell Type Specific

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Faculty Opinions recommendation of Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726681488.793524327 ◽

2016 ◽

Author(s):

Yoshimi Takai

Keyword(s):

Cerebral Cortex ◽

Alternative Splicing ◽

Cell Fate ◽

Cell Type ◽

Specific Alternative ◽

Cell Type Specific

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Neuronal cell type–specific alternative splicing is regulated by the KH domain protein SLM1

The Journal of Cell Biology ◽

10.1083/jcb.201310136 ◽

2014 ◽

Vol 204 (3) ◽

pp. 331-342 ◽

Cited By ~ 53

Author(s):

Takatoshi Iijima ◽

Yoko Iijima ◽

Harald Witte ◽

Peter Scheiffele

Keyword(s):

Alternative Splicing ◽

Neuronal Cell ◽

Specific Gene ◽

Cell Type ◽

Specific Expression ◽

Splicing Regulators ◽

Cell Type Specific Expression ◽

Kh Domain ◽

Cell Type Specific

The unique functional properties and molecular identity of neuronal cell populations rely on cell type–specific gene expression programs. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Neuronal cells exhibit particularly extensive alternative splicing regulation. We report a highly selective expression of the KH domain–containing splicing regulators SLM1 and SLM2 in the mouse brain. Conditional ablation of SLM1 resulted in a severe defect in the neuronal isoform content of the polymorphic synaptic receptors neurexin-1, -2, and -3. Thus, cell type–specific expression of SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo.

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An alternative splicing switch shapes neurexin repertoires in principal neurons versus interneurons in the mouse hippocampus

eLife ◽

10.7554/elife.22757 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 30

Author(s):

Thi-Minh Nguyen ◽

Dietmar Schreiner ◽

Le Xiao ◽

Lisa Traunmüller ◽

Caroline Bornmann ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Network Activity ◽

Cell Type ◽

Splice Isoforms ◽

Mouse Hippocampus ◽

Specific Alternative ◽

Cell Type Specific ◽

Splice Isoform ◽

Functional Features

The unique anatomical and functional features of principal and interneuron populations are critical for the appropriate function of neuronal circuits. Cell type-specific properties are encoded by selective gene expression programs that shape molecular repertoires and synaptic protein complexes. However, the nature of such programs, particularly for post-transcriptional regulation at the level of alternative splicing is only beginning to emerge. We here demonstrate that transcripts encoding the synaptic adhesion molecules neurexin-1,2,3 are commonly expressed in principal cells and interneurons of the mouse hippocampus but undergo highly differential, cell type-specific alternative splicing. Principal cell-specific neurexin splice isoforms depend on the RNA-binding protein Slm2. By contrast, most parvalbumin-positive (PV+) interneurons lack Slm2, express a different neurexin splice isoform and co-express the corresponding splice isoform-specific neurexin ligand Cbln4. Conditional ablation of Nrxn alternative splice insertions selectively in PV+ cells results in elevated hippocampal network activity and impairment in a learning task. Thus, PV-cell-specific alternative splicing of neurexins is critical for neuronal circuit function

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