Alternative splicing as a regulator of development and tissue identity

Abstract

Alternative splicing of eukaryotic transcripts is a mechanism that enables cells to generate vast protein diversity from a limited number of genes. The mechanisms and outcomes of alternative splicing of individual transcripts are relatively well understood, and recent efforts have been directed towards studying splicing networks. It has become apparent that coordinated splicing networks regulate tissue and organ development, and that alternative splicing has important physiological functions in different developmental processes in humans.

Figure 1.. Alternative splicing functions in brain development.

a. Exon 19 (e.19) in Rbfox1 transcript is alternatively spliced giving rise to nuclear and cytoplasmic protein isoforms. In RBFOX-depleted neurons misregulation of splicing networks controlled by RBFOX1 leads to defects in the expression of transcription factors, other splicing factors and synaptic proteins. Introduction of exogenous nuclear RBFOX1 (RBFOX1 containing e.19) rescues changes resulting from the misregulation of splicing networks. Introduction of exogenous cytoplasmic RBFOX1 (lacking e.19) stabilizes multiple mRNAs (for example by interfering with binding of some micro-RNAs (miRNAs)), particularly of synaptic and autism-related genes. This stabilization increases protein expression of the targeted genes rescuing certain neuronal functions. b. During brain development DAB1 protein undergoes a transition from a long to a short isoform, involving skipping of exons 7b and 7c. This developmentally regulated splicing transition of DAB1 is under control of RNA-binding protein NOVA2, which governs exon skipping. DAB1 isoform containing exons 7b and 7c (DAB1–7bc) is more stable and when misexpressed in adults it antagonizes the short variant inducing neuronal migration defects. Consistent with this, Nova2 knock out mice as well as mice in which the DAB1–7bc has been ectopically introduced show defects in neuronal migration, and these defects are rescued by expression of the short DAB1 isoform. c. Alternative splicing of the chromatin remodeler EHMT2/G9a in neuronal differentiation regulates neuronal differentiation. EHMT2 exon 10 inclusion increases during neuronal differentiation and favors EHMT2 nuclear localization. This leads to the increase in histone H3 Lys9 dimethylation (H3K9me2), a histone mark generally associated with transcriptional silencing. In this case, it is plausible to hypothesis that the increase in H3K9me2 might contribute to repression of progenitor-specific genes. Isoform containing exon 10 is thus required for neuronal differentiation through the promotion of exon 10 inclusion on its own pre-mRNA. In consequence, this positive feedback loop reinforces commitment to differentiation. d. Honeybee larvae development into a queen or a worker bee is regulated by DNA-methylation (green pins represent methylation sites), which is influenced by nutrition (larvae developing into queens have lower DNA methylation levels and differentiation into queens can be promoted by silencing DNA-methyl-transferase-3 (DNMT3)). Alternative splicing is more prevalent in methylated genes than in non-methylated ones. In particular, intron between exons 25 and 26 in pre-mRNA of Alk (a metabolic regulator with the capacity to enable growth in a nutrient-independent manner)is differentially methylated between queens and workers. Low methylation correlates with exon 25 inclusion and leads to the development of the larvae as queens.

Figure 2.. Alternative splicing in heart development.

a. The heart is the first organ to form and function during embryogenesis and prenatal heart development has been very well studied at the transcriptional and posttranscriptional levels. Many changes in the heart also occur postnatally, and are associated with the switch between extremely different oxygen and metabolic conditions as well as changes in cardiomyocyte intracellular structure. These physiological and morphological changes are mainly driven by transcriptional and posttranscriptional transitions, including changes in the expression of RNA-binding proteins (RBPs) and splice isoforms. b. During heart development RBM20 regulates splicing of titin – a giant protein able to act as a molecular spring – with exons 50–219 being developmentally regulated^–. In neonates primarily long isoforms (N2BA) are expressed, whereas a shorter isoform (N2B) dominates in adults. The alternatively spliced region of titin is known as the I-band, and controls titin elasticity (with longer regions promoting elasticity). The ratio between N2BA and N2B isoforms thus determines sarcomere length, cardiomyocyte passive tension, and myocardium wallstiffness. RBM20 mutations were identified in humans with cardiomyopathies, linking changes in titin alternative splicing to human pathology^–. FN3: fibronectin type-3 domain, Ig: immunoglobulin-like, PVEK: region rich in proline, glutamate, valine, and lysine.

Figure 3.. Interplay between splicing and epigenetic modifications in spermatogenesis and regulatory feedback loops involving splicing in T-cell activation.

a. Interplay between splicing and epigenetics during spermatogenesis has been demonstrated using a conditional knock out mice lacking MRG15 specifically in postnatal male germ cells. These animals exhibit spermatogenic arrest and sterility. MRG15 depletion triggers intronic segment retentions and one of them occurs in transition nuclear proteins 2 (TNP2). In normal round spermatids MRG15 binds to histone H3 Lys36 trimethylated (H3K36me3) sites between the two TNP2 exons (e.1 and e.2) and it recruits RNA-binding proteins PTBP1 and PTBP2 to these sites, enabling the correct removal of the intron, which generates a functional TNP2 protein. TNP2 and other related proteins then replace histones in chromatin of maturing sperm, and are eventually replaced by protamines, which allow optimal chromatin packing for sperm head elongation observed in mature sperm. When MRG15 is absent PTPB2 is not recruited and the intron is retained leading to a drastic reduction in TNP2 protein levels, and in result arrest of spermatogenesis at a round spermatid stage. Panel is adapted from. b. A positive regulatory feedback loop between JNK signalling and splicing in T-cell activation. This feedback loop comprises the CELF2 splicing factor, which is responsible for alternative splicing of Jun-kinase-kinase MAP-kinase-7 (MKK7). T-cell activation induces CELF2-mediated MKK7 exon 2 (e.2) skipping. This generates a shorter MKK7 isoform that harbours an additional JNK-docking site, which enhances JNK activity and JNK signalling. Enhanced JNK signalling induces CELF2 mRNA stabilization and protein up-regulation, and so CELF2 levels increase with sustained T-cell activation, establishing a positive regulatory feedback loop^,,.