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Intron Retention As a Component of Regulated Gene Expression Programs Hum Genet DOI 10.1007/s00439-017-1791-x REVIEW Intron retention as a component of regulated gene expression programs Aishwarya G. Jacob1 · Christopher W. J. Smith1 Received: 24 February 2017 / Accepted: 29 March 2017 © The Author(s) 2017. This article is an open access publication Abstract Intron retention has long been an exemplar of Introduction regulated splicing with case studies of individual events serving as models that provided key mechanistic insights Alternative splicing (AS) is a widespread process, affect- into the process of splicing control. In organisms such as ing the vast majority of human genes (Barbosa-Morais plants and budding yeast, intron retention is well under- et al. 2012; Merkin et al. 2012). Many alternative splic- stood as a major mechanism of gene expression regulation. ing events (ASEs) are regulated to ensure production of In contrast, in mammalian systems, the extent and func- appropriate protein isoforms in the correct cellular environ- tional signifcance of intron retention have, until recently, ments. Numerous examples of the consequences of ASEs remained greatly underappreciated. Technical challenges upon the function of pairs of protein isoforms have been to the global detection and quantitation of transcripts with well documented (Nilsen and Graveley 2010). Alternative retained introns have often led to intron retention being cassette exons tend to affect intrinsically disordered protein overlooked or dismissed as “noise”. Now, however, with regions, sites of protein–protein interactions, and sites of the wealth of information available from high-throughput post-translational modifcations, and programs of AS have deep sequencing, combined with focused computational the capacity to re-wire protein–protein interaction networks and statistical analyses, we are able to distinguish clear (Buljan et al. 2012; Ellis et al. 2012; Yang et al. 2016). The intron retention patterns in various physiological and path- importance of appropriately regulated AS is underscored ological contexts. Several recent studies have demonstrated by human diseases such as myotonic dystrophy that arise, intron retention as a central component of gene expres- not from aberrant splicing per se, but from mis-regulation sion programs during normal development as well as in of developmental programs of AS, with clinical symptoms response to stress and disease. Furthermore, these studies arising from expression of mRNA isoforms at inappropri- revealed various ways in which intron retention regulates ate stages of development (Cooper et al. 2009). Mis-regu- protein isoform production, RNA stability and translation lation of alternative splicing is also associated with cancers, effciency, and rapid induction of expression via post-tran- and abnormal expression or mutations in splicing factors scriptional splicing of retained introns. In this review, we are known to contribute to tumorigenesis (Anczukow and highlight critical fndings from these transcriptomic stud- Krainer 2016). ies and discuss commonalties in the patterns prevalent in In addition to the widely appreciated role in production intron retention networks at the functional and regulatory of functionally distinct protein isoforms, many regulated, levels. often highly conserved, ASEs generate mRNA isoforms that are channelled to decay pathways such as nonsense mediated decay (AS-NMD) (Ge and Porse 2014; Hillman et al. 2004; Lareau et al. 2007; Lewis et al. 2003; Weis- * Christopher W. J. Smith chenfeldt et al. 2012). Such ASEs are frequently referred [email protected] to as “non-productive” on the basis that one of the RNA 1 Department of Biochemistry, University of Cambridge, isoforms is destined to be degraded rather than translated. Tennis Court Road, Cambridge CB2 1QW, UK However, the “non-productive” label should not be taken 1 3 Hum Genet to imply lack of functionality. In many cases, the ability to 2016). For example, IR has been identifed as a common produce a protein-coding or an NMD-targeted mRNA iso- cause of tumor-suppressor inactivation in cancers (Jung form provides an important regulatory function (McGlincy et al. 2015). and Smith 2008). For example, the transition between Intron retention is most often associated with down- expression of the closely related splicing regulators PTBP1 regulation of gene expression via NMD (IR-NMD) (Ge and and PTBP2, which is important during neuronal differ- Porse 2014) primarily because retained intron sequences entiation, is effected by an AS-NMD event in the PTBP2 that interrupt the main open reading frame (ORF) of the pre-mRNA that is antagonistically regulated by PTBP1 mRNA usually lead to introduction of premature termina- and RBFOX proteins (Boutz et al. 2007; Jangi et al. 2014; tion codons (PTCs). However, this is by no means the only Makeyev et al. 2007). Indeed, the presence of such AS- consequence. The fate of an mRNA with one or more IR NMD events within the pre-mRNAs of splicing regulatory events depends upon a number of factors, including the proteins allows for auto-regulation and cross-regulation location of the IR event within the transcript (Fig. 1): between families of related proteins, as well as control by “master” RBPs, which in turn helps to create robust post- • Nuclear retention and degradation. transcriptional regulatory networks (Jangi and Sharp 2014). • Nuclear retention and storage awaiting signal-induced AS is commonly classifed into seven types of simple splicing. binary events: cassette exons, mutually exclusive exons, • IR in the 5′ UTR can insert an upstream ORF (uORF) alternative 5′ splice sites, alternative 3′ splice sites, intron or other structural features that can activate or repress retention (IR), alternative 3′ terminal exons, and alterna- translational initiation effciency. tive 5′ exons. In addition, many complex ASEs involve • IR in the main ORF can result in PTCs leading to IR- combinations of these simple events (Vaquero-Garcia et al. NMD, or possibly production of truncated proteins. 2016). Of the classes of ASE, IR has probably received • IR in the main ORF can maintain reading frame allow- the least attention in humans and other mammals, at least ing production of pairs of protein isoforms. until recently. This may have resulted in part because of • If the intron is more than ~55 nt into the 3′ UTR, where the diffculty in determining unequivocally that an appar- splicing would lead to NMD, IR can stabilize the RNA ent IR event derives neither from genomic DNA nor from by avoiding NMD. RNA processing intermediates. In contrast to its relatively • IR in the 3′ UTR can introduce cis-elements that affect neglected role in human gene-expression, IR is the most the stability or translational effciency of the mRNA. common type of ASE in plants, fungi, and unicellular eukaryotes, and has consequently long been appreciated as Here, we review progress in understanding the contri- an important regulatory mechanism by researchers using butions of regulated IR in mammalian cells and highlight these model organisms (Pleiss et al. 2007; Syed et al. 2012). examples of its various roles in gene expression modula- Regulated splicing of intron 3 of the Drosophila P-element tion. In particular, we focus on recent transcriptome-wide transposase was one of the earliest examples of cell-type analyses, including those of developmentally regulated specifc AS regulation with clear-cut consequences for the gene expression programs, where IR plays important activity of the encoded protein (Rio et al. 1986). Splicing roles. of P-element intron 3 in germ-cells produces the full length transposase, while retention of intron 3 in somatic cells gives rise to a shorter DNA binding protein that lacks trans- The challenges of detecting and defning intron posase activity and acts as an antagonist of the full-length retention events protein. The P-element transposase also showed how IR can be regulated in a cell-type specifc manner via repres- IR is fundamentally different from other simple ASEs in sors of intron 3 splicing in somatic cells [e.g., (Adams et al. that the sequence of the products of IR is identical to that 1997; Horan et al. 2015; Labourier et al. 2001)]. In view of of genomic DNA and pre-mRNA (at least in the area of this long acknowledged role in many other organisms, the the IR event). This means that precautions need to be taken recent emergence of the varied roles of IR in humans and to ensure that observed IR products are indeed derived other mammals should come as no surprise (Ge and Porse from processed RNA. This involves the use of routine 2014; Wong et al. 2016). Moreover, in addition to physi- controls, such as omission of reverse-transcriptase to rule ologically regulated events, aberrant IR can result from out genomic DNA as the source template, and the use of mutations in splice sites or regulatory sequences. Disease- oligo dT selection of RNA for priming of cDNA synthe- associated mutations in splice sites are most frequently sis to ensure that poly-adenylated RNA is being analyzed. associated with exon skipping (Berget 1995), but in many Use of cytoplasmic polyA RNA can help to reduce the + cases, mutation driven IR can be pathological (Wong et al. signal from nascent RNA, but at the expense of missing 1 3 Hum Genet STOP STOP AUG AUG STOP STOP AAAA Nuclearretained, awaiting signal forsplicing Nuclearretained&degraded Nucleus STOP AUG AAAA STOP AUG STOP AAAA STOP AUG EJC AAAA IR-NMD: PTC in retained intron 3’ UTRIRstabilizesmRNA:NMD Cytoplasm triggers NMD o 2 STOP AUG AUG STOP AAAA AAAA STOP AUG STOP AUG AUG AAAA AAAA uORF 5’UTR IR regulates translation initiation “Exitron” IR encodes protein isoform Fig. 1 Functionally diverse consequences of intron retention. Sche- within the 5′ UTR has the potential to regulate translation initiation in matic illustration of functional consequences of IR. In all cases, the a number of ways, most commonly repressing translation of the main thin black line represents the retained intron.
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