Prospects & Overviews Review essays
Intron retention in mRNA: No longer nonsense Known and putative roles of intron retention in normal and disease biology Justin J.-L. Wong1)2), Amy Y. M. Au1)2), William Ritchie1)2)3) and John E. J. Rasko1)2)4)
Until recently, retention of introns in mature mRNAs has been regarded as a consequence of mis-splicing. Intronretaining transcripts are thought to be non-functional because they are readily degraded by nonsense-mediated decay. However, recent advances in next-generation sequencing technologies have enabled the detection of numerous transcripts that retain introns. As we review herein, intron-retaining mRNAs play an essential conserved role in normal physiology and an emergent role in diverse diseases. Intron retention should no longer be overlooked as a key mechanism that independently reduces gene expression in normal biology. Exploring its contribution to the development and/or maintenance of diseases is of increasing importance.
.
Keywords: alternative splicing; cancer; gene expression; intron retention; miRNAs; non-coding RNAs; nonsensemediated decay
DOI 10.1002/bies.201500117 1)
2) 3)
4)
Gene and Stem Cell Therapy Program, Centenary Institute, Royal Prince Alfred Hospital, Camperdown, Australia Sydney Medical School, University of Sydney, Camperdown, Australia Department of Bioinformatics, Centenary Institute, Royal Prince Alfred Hospital, Camperdown, Australia Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, Australia
These authors contributed equally to this work.
*Corresponding author: John E. J. Rasko E-mail: [email protected] Abbreviations: AS, alternative splicing; IR, intron retention; NMD, nonsense-mediated decay; PTC, premature termination codon; UTR, untranslated region.
Bioessays 38: 41–49, ß 2015 WILEY Periodicals, Inc.
Introduction Although it has been over 3 decades since the astonishing discovery of introns in the laboratories of Phillip Sharp and Richard Robert [1, 2], there is still much to learn concerning their functions. Introns are one of the key features that define eukaryotes, given their absence in prokaryotes [3–5]. As DNA sequences that intervene between adjacent exons, they do not encode protein and hence have been regarded largely as “junk DNA” [6, 7]. They are also considered as a “burden” to an organism because of the time and energy required by the transcriptional and splicing machineries to transcribe and remove long introns in the course of assembling coding sequences for protein production [8]. Malfunction or inefficient splicing of introns can have detrimental effects on cells, thus making eukaryotes potentially vulnerable [9, 10]. The “burden” imposed by introns provokes questions as to their role and raison d’^etre. Nevertheless, it is clear that introns are important for facilitating alternative splicing (AS) so that a single gene can be processed into multiple proteins [11, 12]. This feature is extremely important for promoting protein diversity, which is associated with tissue-specific functions in eukaryotes. Intronic sequences are also known to harbor non-coding RNAs, which regulate distinct biological processes [13, 14]. A surprising burst of discoveries has consolidated the conclusion that many introns are actively retained in mature mRNAs [15–22]. Intron retention (IR) in mRNAs has long been considered a consequence of mis-splicing [8, 23]. Transcripts retaining introns are often removed by nuclear retention and exosome degradation or nonsense-mediated decay (NMD). These mechanisms prevent intron-retaining transcripts from being translated into potentially harmful proteins [23, 24]. Due to the rapidity and efficiency of mRNA decay processes, intron-retaining transcripts are either detected at extremely low levels or not at all using traditional experimental approaches. Using ultra-deep high throughput RNA sequencing coupled with advances in bioinformatic algorithms to detect IR, it has been demonstrated that introns contribute to the regulation of gene expression [18, 19] (Fig. 1A), regulation
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Review essays
J. J.-L. Wong et al.
Prospects & Overviews
....
between adjacent exons by a massive RNAprotein complex known as the spliceosome [35], comprising five RNAs and over 200 proteins [36, 37]. In many cases, selection of specific exonintron splice sites (exon definition) by complexes containing small nuclear ribonucleic proteins (snRNPs) U1, U2, U4/U6, and U5 [38] and auxiliary splicing factors [39] creates alternative splice isoforms. Particularly in cases where intron sizes are 50–55 nt upstream coding regions, because they influence gene expression of an exon–exon junction [44], although intron-retaining differently. The consensus view is that 50 UTR introns are transcripts that escape NMD have been described [45]. important for the regulation of nuclear mRNA export and Experimental approaches designed to inhibit NMD, and cytoplasmic mRNA levels, thereby controlling translation [26, thereby reveal intron-retaining mRNAs otherwise destined 30, 31]. A large number of 30 UTR introns contain premature for degradation, include application of caffeine, emetine, termination codons (PTCs), which elicit NMD to regulate cycloheximide, and overexpression of shRNAs that target the gene expression [32, 33]. The roles of 50 and 30 UTR introns in core NMD factor Upf1/Rent [20, 32, 46]. NMD suppression using regulating gene expression have been reviewed in detail such approaches reveals that normal unmutated genes from elsewhere [34]. Although we have provided examples of UTR diverse pathways, including the cell cycle and splicing, are introns throughout this review by way of comparison, they are subject to NMD [47, 48]. The importance of NMD is emphasized not the main focus of the present work. by the observation that blood stem cells do not survive Here, we review the current understanding of IR with following the loss of NMD [33]. It is therefore conceivable that special attention to introns retained in the coding sequence. splicing, even in normal cells, gives rise to many aberrant We describe the roles of introns that are retained in mature transcripts; the majority presumably being degraded with a mRNAs to regulate normal cellular development in plants and half-life of
Intron retention in mRNA: No longer nonsense Known and putative roles of intron retention in normal and disease biology Justin J.-L. Wong1)2), Amy Y. M. Au1)2), William Ritchie1)2)3) and John E. J. Rasko1)2)4)
Until recently, retention of introns in mature mRNAs has been regarded as a consequence of mis-splicing. Intronretaining transcripts are thought to be non-functional because they are readily degraded by nonsense-mediated decay. However, recent advances in next-generation sequencing technologies have enabled the detection of numerous transcripts that retain introns. As we review herein, intron-retaining mRNAs play an essential conserved role in normal physiology and an emergent role in diverse diseases. Intron retention should no longer be overlooked as a key mechanism that independently reduces gene expression in normal biology. Exploring its contribution to the development and/or maintenance of diseases is of increasing importance.
.
Keywords: alternative splicing; cancer; gene expression; intron retention; miRNAs; non-coding RNAs; nonsensemediated decay
DOI 10.1002/bies.201500117 1)
2) 3)
4)
Gene and Stem Cell Therapy Program, Centenary Institute, Royal Prince Alfred Hospital, Camperdown, Australia Sydney Medical School, University of Sydney, Camperdown, Australia Department of Bioinformatics, Centenary Institute, Royal Prince Alfred Hospital, Camperdown, Australia Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, Australia
These authors contributed equally to this work.
*Corresponding author: John E. J. Rasko E-mail: [email protected] Abbreviations: AS, alternative splicing; IR, intron retention; NMD, nonsense-mediated decay; PTC, premature termination codon; UTR, untranslated region.
Bioessays 38: 41–49, ß 2015 WILEY Periodicals, Inc.
Introduction Although it has been over 3 decades since the astonishing discovery of introns in the laboratories of Phillip Sharp and Richard Robert [1, 2], there is still much to learn concerning their functions. Introns are one of the key features that define eukaryotes, given their absence in prokaryotes [3–5]. As DNA sequences that intervene between adjacent exons, they do not encode protein and hence have been regarded largely as “junk DNA” [6, 7]. They are also considered as a “burden” to an organism because of the time and energy required by the transcriptional and splicing machineries to transcribe and remove long introns in the course of assembling coding sequences for protein production [8]. Malfunction or inefficient splicing of introns can have detrimental effects on cells, thus making eukaryotes potentially vulnerable [9, 10]. The “burden” imposed by introns provokes questions as to their role and raison d’^etre. Nevertheless, it is clear that introns are important for facilitating alternative splicing (AS) so that a single gene can be processed into multiple proteins [11, 12]. This feature is extremely important for promoting protein diversity, which is associated with tissue-specific functions in eukaryotes. Intronic sequences are also known to harbor non-coding RNAs, which regulate distinct biological processes [13, 14]. A surprising burst of discoveries has consolidated the conclusion that many introns are actively retained in mature mRNAs [15–22]. Intron retention (IR) in mRNAs has long been considered a consequence of mis-splicing [8, 23]. Transcripts retaining introns are often removed by nuclear retention and exosome degradation or nonsense-mediated decay (NMD). These mechanisms prevent intron-retaining transcripts from being translated into potentially harmful proteins [23, 24]. Due to the rapidity and efficiency of mRNA decay processes, intron-retaining transcripts are either detected at extremely low levels or not at all using traditional experimental approaches. Using ultra-deep high throughput RNA sequencing coupled with advances in bioinformatic algorithms to detect IR, it has been demonstrated that introns contribute to the regulation of gene expression [18, 19] (Fig. 1A), regulation
www.bioessays-journal.com
41
Review essays
J. J.-L. Wong et al.
Prospects & Overviews
....
between adjacent exons by a massive RNAprotein complex known as the spliceosome [35], comprising five RNAs and over 200 proteins [36, 37]. In many cases, selection of specific exonintron splice sites (exon definition) by complexes containing small nuclear ribonucleic proteins (snRNPs) U1, U2, U4/U6, and U5 [38] and auxiliary splicing factors [39] creates alternative splice isoforms. Particularly in cases where intron sizes are 50–55 nt upstream coding regions, because they influence gene expression of an exon–exon junction [44], although intron-retaining differently. The consensus view is that 50 UTR introns are transcripts that escape NMD have been described [45]. important for the regulation of nuclear mRNA export and Experimental approaches designed to inhibit NMD, and cytoplasmic mRNA levels, thereby controlling translation [26, thereby reveal intron-retaining mRNAs otherwise destined 30, 31]. A large number of 30 UTR introns contain premature for degradation, include application of caffeine, emetine, termination codons (PTCs), which elicit NMD to regulate cycloheximide, and overexpression of shRNAs that target the gene expression [32, 33]. The roles of 50 and 30 UTR introns in core NMD factor Upf1/Rent [20, 32, 46]. NMD suppression using regulating gene expression have been reviewed in detail such approaches reveals that normal unmutated genes from elsewhere [34]. Although we have provided examples of UTR diverse pathways, including the cell cycle and splicing, are introns throughout this review by way of comparison, they are subject to NMD [47, 48]. The importance of NMD is emphasized not the main focus of the present work. by the observation that blood stem cells do not survive Here, we review the current understanding of IR with following the loss of NMD [33]. It is therefore conceivable that special attention to introns retained in the coding sequence. splicing, even in normal cells, gives rise to many aberrant We describe the roles of introns that are retained in mature transcripts; the majority presumably being degraded with a mRNAs to regulate normal cellular development in plants and half-life of
