It is not just about transcription: involvement of brain RNA splicing in substance use disorders

Alternative splicing is a crucial co-transcriptional process that allows for a single gene to produce multiple protein isoforms with distinct functional properties. This phenomenon plays a significant role in regulating transcription and increasing protein diversity in various tissues and cell types. Evidence from many species indicates that substance use can lead to alternatively spliced transcripts in different brain regions, contributing to the complex molecular changes associated with the transition to substance misuse. This review explores the relationship between alternative splicing and SUDs, shedding light on the molecular mechanisms of drug-induced changes in the brain that contribute to these disorders. The exploration of drug-induced changes in alternative splicing represents a recent and evolving field of study. The primary goal of this review is to provide a summary of the literature in this burgeoning research area and highlight new technologies that have expanded our knowledge of mechanisms involved in alternative splicing.

Most genes in higher eukaryotes are transcribed as pre-messenger RNA (pre-mRNA), which undergoes splicing as it is transcribed from the DNA template. The process of RNA splicing involves the removal of introns and the ligation of exons to form mature mRNA (Wilkinson et al. 2020). RNA splicing is enabled by the spliceosome, a megadalton machine composed of uridine-rich small nuclear RNA (snRNA: U1, U2, U4, U5, and U6), proteins from the NineTeen complex (NTC), the NTC-related complex (NTR), RNA binding proteins (RBPs) and RNA-dependent ATPase/helicases (Wilkinson et al. 2020; Ule and Blencowe 2019). RBPs in the spliceosome are commonly referred to as splicing factors and play a pivotal role in determining the specificity of splice site selection and the formation of the exon definition complex (the complex of RNA binding proteins and regulatory elements that define exon boundaries) by recognizing specific sequences within the precursor mRNA. These sequences are known as splicing enhancers or silencers. RBPs bound to splicing enhancers stabilize the assembly of the spliceosome and promote exon splicing (Wan et al. 2019; Matera and Wang 2014). Conversely, when RBPs bind to splicing silencers, they impede the assembly of the spliceosome, leading to the inhibition of exon definition complex formation.

Alternative splicing involves the use of alternative splice sites to join exons in various combinations, thereby generating multiple transcripts from a single gene. Alternatively, spliced transcripts can generate diverse protein isoforms with distinct cellular functions through the inclusion of alternative functional domains (Sibley et al. 2016). Alternative splicing results in events such as exon skipping, in which an exon is excluded in the mature mRNA (Wan et al. 2019), the use of mutually exclusive exons, in which only one exon from a cluster is included, intron retention, in which the intron is not spliced out (Galante et al. 2004), and the use of alternative 3′ and 5′ splice sites located within an intron or exon (Blencowe 2006). Alternative splicing is a fundamental cellular process with dual implications for health, serving as a crucial mechanism for normal physiological functions but also playing a role in the pathogenesis of disease. Alternative splicing enhances genomic and protein diversity, which is essential for tissue-specific functions and overall cellular homeostasis. For instance, in the nervous system, alternative splicing ensures proper neuronal development, neuronal migration, axon guidance, and synaptogenesis (Zhou et al. 2018; Raj and Blencowe 2015). However, when alternative splicing becomes dysregulated, it may lead to the production of dysfunctional proteins or disrupt vital cellular processes, contributing to the onset and progression of neurological and psychiatric diseases (Nik and Bowman 2019). Studies indicate that exposure to drugs of abuse results in alternatively spliced transcripts in different brain regions, potentially contributing to the development of SUDs (Van Booven et al. 2021; Xu et al. 2014, 2021; Piltonen et al. 2021; Huggett et al. 2022; Li et al. 2023; Krapacher et al. 2022).

In recent years, researchers have been exploring how repeated drug exposure can influence splicing patterns and how these alterations might contribute to the physiological and behavioral effects associated with drug use. Table 1 lists studies on drug-induced changes in RNA splicing. One way in which drugs of abuse can impact RNA splicing is by altering the expression of genes encoding splicing factors (Van Booven et al. 2021; Carvalho et al. 2023). Psychotropic drugs exert their effects by targeting synaptic elements, ion channels, and neurotransmitter receptors, subsequently modifying intracellular signaling cascades (Nestler and Lüscher 2019; Robison and Nestler 2011). The activation or inhibition of these specific signaling pathways, in turn, leads to the upregulation or downregulation of genes encoding splicing factors. Altered protein levels of splicing factors can then exert regulatory control over alternative splicing in genes responsible for encoding neurotransmitter receptors, transporters, and other essential proteins involved in synaptic transmission or glial cell function, ultimately contributing to drug-related changes in neuronal signaling and function, and behavior (Fig. 1).

It has now become recognized that since splicing is a co-transcriptional process, chromatin can significantly impact the final splicing outcome (Agirre et al. 2021). The kinetic model suggests that chromatin states can slow down RNA polymerase II, thus increasing the time during which splicing regulators can bind to nascent RNA (Agirre et al. 2021). The recruitment model proposes that chromatin modifications, such as histone modifications and DNA methylation, can modulate the binding of splicing factors to pre-mRNA by recruiting chromatin-binding proteins that serve as adaptors between the chromatin and the splicing machinery (Luco et al. 2011). Drugs of abuse, such as cocaine and alcohol alter histone modifications and DNA methylation in key brain regions by altering the expression of histone- and DNA-modifying enzymes and metabolites required for histone acetylation and DNA methylation (Walker et al. 2015; Pandey et al. 2008; Feng et al. 2014; Lev Maor et al. 2015; Mews et al. 2019; Gatta et al. 2017). Therefore, another way in which drugs of abuse can potentially impact splicing is through the changes in epigenetic modifications at specific genes (Xu et al. 2021; Kyzar et al. 2019).

Epigenetic modifications can influence alternative splicing through various mechanisms. For instance, DNA methylation can impact splicing through promoter and intragenic methylation, influencing the recruitment of splicing regulators (Maunakea et al. 2013; Yearim et al. 2015). Histone modifications, such as acetylation, methylation, and ubiquitination, affect splicing by influencing splice site selection (Rahhal and Seto 2019; Hu et al. 2017). Chromatin remodeling complexes can directly alter splice site accessibility (Casteels et al. 2022). Non-coding RNAs, like microRNAs and lncRNAs, can act as splicing regulators or compete with splicing factors (Statello et al. 2021; Romero-Barrios et al. 2018; Stanek 2021). RNA modifications, such as N6-Methyladenosine (m6A), can influence splicing by recruiting or repelling splicing factors (Yang et al. 2018; Wang et al. 2022).

Feng et al. (2014) had previously examined combinations of histone modifications associated with transcript variant expression and found specific “chromatin signatures” correlated with cocaine-induced alternative splicing in the nucleus accumbens (NAc), a crucial brain region associated with cocaine-reward behavior. A subsequent study conducted in mice reported that cocaine injections induced changes in the enrichment of specific histone modifications in relation to various types of alternatively spliced exons within the nucleus accumbens (NAc) (Hu et al. 2017). Specifically, cocaine led to genome-wide differences in the enrichment of histone H3 lysine 36 trimethylation (H3K36me3), histone H3 lysine 27 trimethylation (H3K27me3), histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 4 monomethylation (H3K4me1). Notably, H3K36me3 enrichment showed the strongest association with alternative splicing and served as a robust predictor for exon exclusion (Hu et al. 2017).

In another study, it was observed that both cocaine self-administration and the overexpression of SET2, a histone methyltransferase catalyzing the addition of methyl groups to H3K36, resulted in an overlap of alternative exons enriched for H3K36me3 and containing the binding motif for Srsf11, a serine and arginine-rich splicing factor 11 (Xu et al. 2021). Interestingly, the authors found that Srsf11 mRNA itself underwent differential splicing and was enriched in H3K36me3 in mice treated with cocaine or SET2 overexpression. Targeted enrichment of H3K36me3 at Srsf11 was achieved using a CRISPR-Cas9 method, by expressing nuclease-deficient Cas9 fused to the histone methyltransferase SET2 (dCas9-SET2) and Srsf11 single guide (sg)RNA in the NAc. This replicated the cocaine-induced alternative splicing patterns and enhanced cocaine-reward behavior. These findings highlight the intricate relationship between drug-induced epigenetic modifications, their impact on splicing processes, and the subsequent effects on drug-related behavior.

While it has been established that alcohol exposure triggers changes in histone modifications and DNA methylation, the specific relationship of these epigenetic alterations to alcohol-induced alternative splicing remains to be determined (Pandey et al. 2008; Palmisano and Pandey 2017; Berkel and Pandey 2017). Similarly, it is known that chronic alcohol consumption alters the RNA methylome (m6A modification) (Liu and Zhang 2022), however, the contribution of this modification to changes in splicing and drug-related behavior remains to be investigated. Epigenetic marks can crosstalk, modulate transcription factor binding, alter 3D chromatin structure, exhibit cell-type-specific patterns, and collectively shaping context-dependent alternative splicing. Thus, this field of research is critical for understanding the complexity of gene regulation and its implications for behaviors related to addiction.

Recent studies have highlighted that changes induced by drugs of abuse occur not only at the transcriptional level but also during RNA splicing. Current literature suggests that the impact of drugs of abuse on splicing can vary depending on factors such as the specific drug used, the duration and pattern of use, and individual genetics. Despite the progress made, numerous avenues for future exploration remain open. While genome-wide studies provide an overview of the components of the spliceosome altered due to drug exposure, there is a need for a deeper understanding of the precise molecular mechanisms through which drugs of abuse influence the splicing machinery and splicing factor expression. Furthermore, it is necessary to elucidate the functional consequences of drug-induced splicing changes for synaptic function and behavior. This can be achieved by knocking down targets via RNA interference (RNAi) or CRISPR/Cas9 methods followed by behavioral tests. Questions arise regarding whether alternative splicing contributes to drug-seeking behavior or anxiety and depression during drug withdrawal. Additionally, there is a need to examine the differences between acute and chronic effects of drug-induced splicing alterations. Understanding the consequences of drug exposure during developmental periods, such as adolescence, on alternative splicing in adulthood is essential. Moreover, exploring whether exposure to drugs of abuse leads to differential RNA splicing between males and females and whether this sex difference influences the risk of developing a SUD remains to be investigated.

Methods to study RNA splicing have undergone a transformative evolution. In the past, classical techniques primarily relied on PCR/RT-PCR and gel electrophoresis to analyze splicing patterns, often limiting the simultaneous examination of multiple splicing events. However, the emergence of high-throughput sequencing technologies, such as RNA-Seq, allows for a comprehensive and genome-wide analysis of alternative splicing events. Moreover, the recent integration of long-read sequencing technologies generates full-length transcripts, overcoming the challenges posed by short-read technologies in accurately identifying transcript isoforms (Leung et al. 2021; Pepke et al. 2009). Chromatin immunoprecipitation followed by sequencing (Chip-Seq), allows the investigation of the physical interactions between splicing regulators and chromatin regions associated with specific splicing events (Busch et al. 2020). Finally, RNA binding assays, such as RNA immunoprecipitation (RIP) or cross-linking immunoprecipitation (CLIP), followed by sequencing, allow researchers to identify the RNA molecules directly bound by splicing regulators at the whole transcriptome level. We predict that our knowledge of alternative splicing changes induced by drug exposure, and the behavioral consequences of these changes, will rapidly increase in the next several years.