Alternative Splicing in Nervous System

What is Alternative Splicing?

Alternative splicing is a versatile form of genetic control to process a pre-mRNA into multiple mRNA isoforms, differing in the precise combination of exon sequences. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome and expand proteomic complexity and play numerous essential roles in gene regulation. Alternative splicing regulates the localization of proteins, their enzymatic properties, and their interaction with ligands. Alternative splicing is also a key process to increased proteomic and functional complexity and is prevalent in the human nervous system. Moreover, numbers of specialized cell types and activities, such as the mammalian brain, undergo relatively frequent alternative splicing.

Fig.1 Two forms of alternative splicing. (Lipscombe, 2005)

Roles of Alternative Splicing in Nervous System

Alternative splicing has emerged as a critical mechanism of functional regulation in the human genome, and many types of alternative splicing have been conserved and subject to cell-, tissue-, or developmental-specific regulation. Remarkably, high-throughput transcriptomic profiling approaches have revealed that alternative splicing of precursor mRNAs is particularly widespread in the nervous system. In nervous systems, alternative splicing has emerged as a fundamental mechanism for diversifying protein isoforms and the spatiotemporal control of transcripts.

In recent years, fundamental roles for alternative splicing in neural development and neuronal networks establishment and function have become increasingly evident. Alternative splicing has played a significant role in the evolutionary expansion of proteomics and functional complexity in the nervous system, with emerging key roles in synaptogenesis, neuronal growth, neurite outgrowth, axon guidance, neuronal network function, ion channel activity, and long-term potentiation. Other data support the strong association of alternative splicing and nervous system functions. Genomics studies of alternative splicing tissue specificity found that the largest group of tissue-specific alternative splice forms was detected in brain, retina, and nerve-derived tissue sources.

Fig.2 Examples of contributions of alternative splicing regulation to neural development and plasticity. (Furlanis, 2018)

Factors Regulating Alternative Splicing

The major splicing factors that regulate constitutive and alternative splicing include the heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins. These splicing factors have been identified that show cell- or tissue-specific or enriched expression. Most of these proteins are enriched in neural and/or muscle tissues. In some cases, tissue-specific alternative splicing events could be regulated by different combinations of widely expressed factors and, in other cases, by cell/tissue-specific factors.

These proteins, including Nova AS regulators (Nova-1/2), nPTB/BrPTB, and members of the CELF/Bruno-like, Elav, Fox, and Muscleblind families of RNA binding proteins, have participated in differential alternative splicing regulation in the nervous system or other tissues. The binding of cell/tissue-specific factors to these cis-acting elements affects splice site choice by various specific mechanisms that generally result in the promotion or disruption of interactions of splicing components during the early stages of spliceosome formation.

Alternative Splicing and Neurological Diseases

Alternative splicing to gene expression in the nervous system is important because splicing misregulation or other errors of RNA metabolism cause several forms of neurological diseases. The best-characterized neurological diseases associated with alternative splicing are inherited frontotemporal dementia and parkinsonism linked to chromosome 17 and spinal muscular atrophy resulting from mutations in the survival of motor neurons (SMN) gene. In both cases, splicing defects of identified exons have been associated with the disease phenotype. Others such as spinocerebellar ataxia 8 and amyotrophic lateral sclerosis are also related to abnormal alternative splicing. Thus, the first splicing therapeutics are beginning to be developed. In rodent models of spinal muscular atrophy, antisense oligonucleotides and small molecules that modify the splicing of the endogenous SMN2 gene recover SMN2 splicing activity and provide significant benefits to patients.

Table.1 Cellular processes are changed by alternative splicing in the nervous system. (Kelemen, 2013)

Tools for Analyzing Alternative Splicing in Nervous System

Experimental methods for studying alternative splice forms are in the quick update. The coupling of various computational and experimental approaches advanced our understanding of the mechanisms and functions of alternative splicing significantly. Traditional methods, including Northern or Western blots complemented by RT-PCR, probes the specific difference between one transcript and another. However, a limitation of current investigations of neural alternative splicing regulation is the scarcity of convenient tools to monitor and manipulate isoform expression at the single-cell level. This can be resolved at least part of the challenge with the advent of single-cell sequencing methods.

Single-cell sequencing can be used to detect additional examples of alternative splicing events that are differentially regulated between individual neurons. It can also identify cell-specific alternative splicing regulators based on the differences in expression levels. In addition, other methods such as translating ribosome affinity purification (TRAP) can determine the translation profile of individual cell types and detect cell-specific alternative splicing factors and protein types. These methods will become powerful tools for future investigations into cell-type-specific alternative splicing combined with single-neuron imaging techniques.

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References

1. Lipscombe, D. Neuronal proteins custom designed by alternative splicing. Current opinion in neurobiology. 2005, 15(3), 358-363.
2. Furlanis, E.; Scheiffele, P. Regulation of neuronal differentiation, function, and plasticity by alternative splicing. Annual review of cell and developmental biology. 2018, 34, 451-469.
3. Kelemen, O.; et al. Function of alternative splicing. Gene. 2013, 514(1), 1-30.