Control of alternative splicing in immune responses: many regulators, many predictions, much still to learn

Abstract

Most mammalian pre-mRNAs are alternatively spliced in a manner that alters the resulting open reading frame. Consequently, alternative pre-mRNA splicing provides an important RNA-based layer of protein regulation and cellular function. The ubiquitous nature of alternative splicing coupled with the advent of technologies that allow global interrogation of the transcriptome have led to an increasing awareness of the possibility that widespread changes in splicing patterns contribute to lymphocyte function during an immune response. Indeed, a few notable examples of alternative splicing have clearly been demonstrated to regulate T-cell responses to antigen. Moreover, several proteins key to the regulation of splicing in T cells have recently been identified. However, much remains to be done to truly identify the spectrum of genes that are regulated at the level of splicing in immune cells and to determine how many of these are controlled by currently known factors and pathways versus unknown mechanisms. Here, we describe the proteins, pathways, and mechanisms that have been shown to regulate alternative splicing in human T cells and discuss what is and is not known about the genes regulated by such factors. Finally, we highlight unifying themes with regards to the mechanisms and consequences of alternative splicing in the adaptive immune system and give our view of important directions for future studies.

Fig. 1. Common patterns of AS

Rectangles represent exons and lines represent introns. Dotted lines connect exons that are joined together to form the final mRNA. Red and blue regions are those that can be differentially included in the mature mRNA. Dotted lines above and below represent the two alternate possible splicing patterns.

Fig. 2. General mechanism of splicing and its regulation

(A) Spliceosome assembly. The 5′ end of introns are defined by the 5′ splice site (GUAAGU), and the 3′ end of introns are defined by the branch point sequence (BPS), polypirimidine tract (PPT), and the 3′ splice site AG dinucleotides. The U1, 2, 4, 5 and 6 represent the snRNPs which assemble with substrate and each other as shown. The NTC is an additional snRNA-free spliceosomal subunit. For more details see Motta-Mena et al. (58). (B) Regulation of alternative splicing. Enhancer auxiliary elements are denoted in green for exonic (ESE) or intronic (ISE) splicing enhancers. Silencer auxiliary elements are denoted in red for exonic (ESS) or intronic (ISS) splicing silencers. The activities of these auxiliary elements are often mediated through binding of SR and hnRNPs, two common families of proteins described in the text, among other RNA binding proteins.

Fig. 3. Splicing factors known to regulate AS in immune responses

Domain schematics for each factor are displayed. RRM, RNA recognition motif;φRRM, pseudo RRM; G, glycine-rich region; P, proline-rich region; RGG, arginine/glycine/glycine repeat region; P, Q, proline/glutamine rich; RS, arginine/serine-rich; KH – K homology domain. Binding preferences for each factor are specified for hnRNP L (66), hnRNP LL (64), PSF (75), CELF2 (78), TIA-1 (85), SRSF1(115), and Sam68 (97).

Fig. 4. Mechanism for the regulation of CD45 alternative splicing

The CD45 ARS is depicted in orange/red and the ESE in green. In resting cells, hnRNP L (L) regulates basal levels of exon 4 repression, while SRSF1 mediates basal levels of exon 5 inclusion. Exon 5 inclusion is further regulated at the level of transcription. CTCF binds to the exon 5 ESE at the DNA level and promotes Pol II pausing around that region to promote exon 5 inclusion. Upon activation hnRNP LL and PSF join hnRNP L at the exon 4 and 6 ARSs to promote further repression, while only hnRNP L and PSF associates to exon 5.

Fig. 5. Networks of signaling pathways that regulate AS in T cells

Summary of several known kinases (blue/purple diamonds) and RNA binding factors (orange/red ovals) that have been shown to be regulated in response to antigen (Ag) stimulation or Fas Ligand (FasL) signaling in T cells, and the genes (text) that they regulate. Solid arrows and lines indicate direct activities, dotted arrows/lines indicate known relationships that are likely indirect. ‘other’ indicates the fact that there are likely many other signaling pathways that are also involved in mediating antigen-induced changes in AS that are yet to be discovered. Question marks are meant to emphasize that although each signaling pathway so far has only been linked to regulation of 1–2 genes, each branch likely regulates a program of co-regulated genes. Cross-coordination between branches is also anticipated, as observed for CD45. See text for detailed description of each pathway.