non-coding RNA Review Epigenetic Regulation of Alternative Splicing: How LncRNAs Tailor the Message Giuseppina Pisignano 1,* and Michael Ladomery 2,* 1 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK 2 Faculty of Health and Applied Sciences, University of the West of England, Coldharbour Lane, Frenchay, Bristol BS16 1QY, UK * Correspondence: [email protected] (G.P.); [email protected] (M.L.) Abstract: Alternative splicing is a highly fine-tuned regulated process and one of the main drivers of proteomic diversity across eukaryotes. The vast majority of human multi-exon genes is alternatively spliced in a cell type- and tissue-specific manner, and defects in alternative splicing can dramatically alter RNA and protein functions and lead to disease. The eukaryotic genome is also intensively transcribed into long and short non-coding RNAs which account for up to 90% of the entire tran- scriptome. Over the years, lncRNAs have received considerable attention as important players in the regulation of cellular processes including alternative splicing. In this review, we focus on recent discoveries that show how lncRNAs contribute significantly to the regulation of alternative splicing and explore how they are able to shape the expression of a diverse set of splice isoforms through several mechanisms. With the increasing number of lncRNAs being discovered and characterized, the contribution of lncRNAs to the regulation of alternative splicing is likely to grow significantly. Keywords: long non-coding RNAs; alternative splicing; splicing factors; post-transcriptional regulation Citation: Pisignano, G.; Ladomery, 1. Introduction M. Epigenetic Regulation of In the late 1970s, researchers were interested in gaining a better understanding of Alternative Splicing: How LncRNAs the mechanisms of adenoviral gene expression when they noticed something unusual, Tailor the Message. Non-coding RNA a long adenoviral transcript hybridized to the viral genome forming a three-stranded 2021 7 , , 21. https://doi.org/10.3390/ mRNA:DNA hybrid structure with an intervening DNA sequence that did not match the ncrna7010021 mature mRNA [1]. It then became apparent that these intervening sequences were in fact introns and that the primary transcript is made by a succession of exonic and intronic Received: 21 January 2021 sequences. In what is now thought to be a mainly co-transcriptional process, introns are Accepted: 22 February 2021 Published: 11 March 2021 ‘spliced’ out from the mRNA precursor (pre-mRNA) and the exons joined together through two transesterification reactions catalysed by a complex molecular machinery consisting of Publisher’s Note: MDPI stays neutral five small nuclear ribonucleoproteins (snRNPs), called the spliceosome [2]. Over the past with regard to jurisdictional claims in decades it has become clear that pre-mRNA splicing is a widespread phenomenon across published maps and institutional affil- eukaryotes and that a single gene can generate multiple transcripts often encoding different iations. proteins by a process known as alternative splicing (AS) [3]. Many types of AS are possible, including “cassette exons”, “alternative 50 and 30 splice sites”, “alternative first exons” (through different promoters), “alternative last exons” (through different polyadenylation sites), “mutually exclusive exons” and “retained introns” [4]. Fundamental to the process of AS is the definition of the precise location of 50 (donor) and 30 (acceptor) splice sites and the Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. assembly of the spliceosome complex. The first relies on splicing factors (SFs), a category of This article is an open access article RNA-binding proteins (RBPs) expressed in a tissue and stage-specific way that recognize distributed under the terms and regulatory elements within exons and introns. Importantly, SF activity is in turn modified conditions of the Creative Commons by splicing factor kinases and phosphatases activated through cell signaling mechanisms. Attribution (CC BY) license (https:// AS greatly enhances proteome diversity and represents an essential aspect of gene creativecommons.org/licenses/by/ expression in development, normal physiology and disease across eukaryotes [5], from 4.0/). single-celled yeast to humans [6]. The advent of high-throughput sequencing technologies Non-coding RNA 2021, 7, 21. https://doi.org/10.3390/ncrna7010021 https://www.mdpi.com/journal/ncrna Non-Coding RNA 2021, 7, 2 of 20 in a tissue and stage-specific way that recognize regulatory elements within exons and introns. Importantly, SF activity is in turn modified by splicing factor kinases and Non-coding RNA 2021, 7, 21 2 of 19 phosphatases activated through cell signaling mechanisms. AS greatly enhances proteome diversity and represents an essential aspect of gene expression in development, normal physiology and disease across eukaryotes [5], from revealedsingle-celled that yeast ~92–94% to humans of human [6]. The multi-exon advent of genes high-throughput are alternatively sequencing spliced [technologies7], increasing therevealed interest that in ~92–94% understanding of human the multi-exon mechanisms genes underpinning are alternatively its spliced regulation. [7], increasing With the discoverythe interest and in growingunderstanding importance the mechanis of non-codingms underpinning RNAs, the nature its re ofgulation. AS regulation With the has becomediscovery more and complex.growing importance Both short of (<200 non-codi nt) andng RNAs, long (>200 the nature nt) non-coding of AS regulation RNAs has can contributebecome more to the complex. regulation Both of short AS in (<200 many nt different) and long ways; (>200 either nt) indirectlynon-coding by RNAs regulating can thecontribute activity to of the splice regulation factors; of or AS directly, in many by interactingdifferent ways; with either pre-mRNAs. indirectly Long by regulating non-coding RNAsthe activity (lncRNAs) of splice are factors; particularly or directly, well by suited interacting to these with roles pre-mRNAs. due to their Long demonstrated non-coding capacityRNAs (lncRNAs) to act as regulatory are particularly molecules well that suited modulate to these gene roles expression due to their at every demonstrated level. Either alone,capacity or into associationact as regulatory with partner molecules proteins, that modulate these long gene RNA expression polymerase at IIevery transcripts level. haveEither been alone, shown or in to association take part inwith a wide part rangener proteins, of developmental these long processesRNA polymerase and disease II intranscripts complex have organisms been shown [8–10 ].to take Here, part we in review a wide the range current of developmental knowledge ofprocesses the multiple and mechanismsdisease in complex through organisms which lncRNAs [8–10]. contributeHere, we review to the regulationthe current of knowledge AS (Figure of1 andthe Tablemultiple1). mechanisms through which lncRNAs contribute to the regulation of AS (Figure 1 and Table 1). FigureFigure 1. 1. RegulationRegulation of pre-mRNA pre-mRNA splicing splicing by by lncRNAs. lncRNAs. LncRNA LncRNAss (red) (red) are areable able to control to control pre-mRNA pre-mRNA splicing splicing by (a by) (amodifying) modifying chromatin chromatin accessibility accessibility through through recruiting recruiting or orim impedingpeding access access to to chromatin chromatin modifying modifying complexes complexes at at the the transcribedtranscribed genomic genomic locus. locus. InIn somesome cases,cases, thisthis mightmight resultresult in more drastic long-range long-range structural structural changes; changes; (b (b) )interacting interacting withwith the the transcribed transcribed genomicgenomic locuslocus throughthrough anan RNA-DNARNA-DNA hybrid; ( c) hybridizing with with the the pre-mRNA pre-mRNA molecule molecule (light (light blue); (d) promoting SF recruitment or by sequestering SFs into specific subnuclear compartments, thereby interfering blue); (d) promoting SF recruitment or by sequestering SFs into specific subnuclear compartments, thereby interfering with with SF activities. SF activities. 2. LncRNAs Regulate Alternative Splicing through Chromatin Modification The eukaryotic genome is tightly packaged into chromatin fibers consisting of DNA wrapped around nucleosomes made of histone proteins. Post-translational modifications (PTMs), such as methylation, acetylation, phosphorylation, and ubiquitination, occur on the histone tails that are functionally linked to the epigenetic regulation of gene expression. By defining the accessibility to chromatin, histone modifications demarcate amenable or silenced chromatin domains which ultimately reflect the activity of gene transcription. An intimate relationship exists between lncRNAs and chromatin conformation [11,12]. LncRNAs regulate chromatin modifications by recruiting or directly interacting with Non-coding RNA 2021, 7, 21 3 of 19 histone-modifying complexes or enzymes at specific chromosomal loci; these in turn modulate gene transcription [13–18]. Histone modification signatures can also influence AS through a chromatin-reading protein which acts as an adaptor linker between the RNA polymerase II (RNAPII) and the pre-mRNA splicing machinery [19]. Several studies have also demonstrated that the local chromatin context influences the RNAPII elongation rate which in turn affects AS [20–22]. A possible lncRNA-mediated crosstalk between histone modifications