Alternative Splicing in Innate Antiviral Immunity

Recent genome and transcriptome analyses have shown that coding genes amount to just a fraction of the entire genome. One of the mechanisms that cells use to introduce diversity to the proteome is through alternative splicing of genes. Using new RNA sequencing technologies, studies have shown that approximately 90% of genes produce alternative splice variants. Defining how alternative splicing regulates interferon and cytokine actions is an emerging and highly important research area.

Splicing changes occur within the coding and nontranslated regions of the mRNA. As these data emerge, we have been left with the challenge of understanding the biological consequences of alternative splicing on the cell. Alternative splice forms can be produced by, but not limited to, skipping or inclusion of exons by splicing of premature-messenger RNA (mRNA), employing alternative transcription start sites, or alternative polyadenylation. Alternative splicing can also result in the inclusion or skipping of exons, retention of introns, and the use of variable 5′ and 3′ untranslated regions (UTRs). These changes can lead to insertions, deletions, or truncations of the open reading frame, resulting in either gain or loss of function of the encoded protein. While some alternative splice forms that retain introns could be targeted for nonsense-mediated decay, others might include regulatory elements in the 3′ or 5′ UTRs to induce mRNA decay or suppress translation. As one could predict, aberrant changes in splicing could have severe implications in immune homeostasis and response against pathogens. As we mark 50 years since the discovery of splicing and more recent crystallization of the spliceosome complex, it is imperative that we extend our basic knowledge of splicing into understanding the complexity of alternative splicing in the immune system.

For an exon to be included in the mRNA, the spliceosome complex has to bind to its splice sites. Subsequent inclusion of the exon depends on several factors that are encoded in the gene, including the strength of the splice sites and neighboring enhancer or suppressor sequences that facilitate splicing. These interactions determine the expression of the primary or alternatively spliced mature mRNA and protein. However, there are numerous RNA-binding proteins that bind to splicing sites that could potentially affect alternative splicing. The regulatory motifs and spliceosome machinery are ancient and conserved across species. However, it remains to be seen if any of these proteins are modulated by microbes and the host for their survival, and perhaps more interestingly, whether single nucleotide polymorphisms in the regulatory motifs or the spliceosome machinery influence disease susceptibility. Although most of the work on alternative splicing has focused on development and cancer, defining these mechanisms will have direct implications for understanding how alternative splicing impacts immunity.

One of the early roles of alternative splicing affecting immunity was discovered in genes that are involved in antigen presentation and splicing of IgM and IgD immunoglobulin isotypes. The production of soluble and membrane-bound forms of IgM and IgD is also controlled by alternative splicing. Moreover, interferons and cytokines that express alternative isoforms display altered properties of mRNA stability, protein stability, glycosylation, secretion, or activity through receptor–ligand interactions that impart distinct biological actions. Some cytokine receptors are known to be alternatively spliced to downregulate surface expression or act as receptor antagonists to dampen cytokine signaling. Additionally, aberrant changes in splicing of the ligands or the receptors of cytokines have been shown to have pathophysiological effects.

Hundreds of effector genes are induced by interferons, interleukins, and chemokines, and the functional activity of these genes has been an area of significant research interest in the interferon and cytokine research community. Of specific interest are the interferon-induced genes (ISGs) that have been classified as broadly antiviral. Viruses are known to antagonize ISGs while the host remodels its gene expression programs to neutralize viral infection, making this an intriguing interface for host–pathogen interactions and survival. Emerging evidence suggests that alternative splicing of ISGs could be one such innovation where the host alters the localization, function, and activity of ISGs to neutralize the virus. It is important to investigate the various mechanisms through which viruses hijack the cellular alternative splicing machinery to their advantage. Therefore, identifying new or altered functions of alternatively spliced ISGs in the context of interferon responses and viral infections are warranted.

mRNA stability and protein output are influenced by the regulatory regions in the 3′ and 5′ UTRs. These regulatory regions interact with RNA-binding proteins (RBPs), microRNAs, and long noncoding RNAs that can affect the stability of the mRNA. Alternative splicing can modulate mRNA stability and translation potential of a gene through variations in the length and sequences of regulatory regions. Temporal changes in the UTRs mediated by alternative splicing would provide a rheostat for initiation and resolution of immune responses during infection. Any aberrant changes that affect the splicing could pose severe consequences to immune homeostasis or affect viral clearance.

Tissue-specific expression of alternative splice variants suggests that there might be differences in the expression of proteins that are required for inducing alternative splicing. With advances in single-cell sequencing, it is now possible to profile cell-specific splicing and differential expression of RBPs during infection. CRISPR-mediated gene editing technologies will help to identify RBPs essential for alternative splicing that could be modulated during viral infection or subsequent interferon responses. As immune genes are under continuous positive selection, the sequence identities are low across vertebrate species, even though the functions remain largely conserved. As the sequence conservation is lower in both coding and non-coding regions of the gene, it is likely that sequence differences have an effect on the complexity of splicing across species. Although mice have served as excellent models for studying the immune system, we should be aware that alternatively spliced genes may not be fully conserved between humans and mice. Documenting alternative splice forms, sequence motifs, RBPs, and their function will add an additional layer of understanding and complexity to the regulation of immune responses. Single nucleotide polymorphisms have known effects on splicing. Therefore, production of a cell-specific global alternative splicing map will aid in predicting the effects of polymorphisms in viral resistance or susceptibility.