Insights & Perspectives Think again Introns in UTRs: Why we should stop ignoring them Alicia A. Bicknell1)y, Can Cenik1)y, Hon N. Chua2), Frederick P. Roth2) and Melissa J. Moore1)3)* Although introns in 50- and 30-untranslated regions (UTRs) are found in many considered as distinctive entities with protein coding genes, rarely are they considered distinctive entities with specific regulatory functions. Indeed, until very recently the prevailing view specific functions. Indeed, mammalian transcripts with 30-UTR introns of 50-UTR introns (5UIs) was that they are often assumed nonfunctional because they are subject to elimination by are only special insofar as they are prox- nonsense-mediated decay (NMD). Nonetheless, recent findings indicate that imal to the 50 end of the transcript. 50- and 30-UTR intron status is of significant functional consequence for the Further, a common view of 30-UTR regulation of mammalian genes. Therefore these features should be ignored introns (3UIs) is that they are signatures no longer. of nonfunctional transcripts arising solely from genomic noise (e.g. pseudo- genes, transposons), genetic mutation, Keywords: or errors in splicing. This view stems ALREX; intron; nonsense-mediated decay; 30-UTR; 50-UTR . from the observation that mammalian mRNAs with an intron excision site >55 nucleotides downstream of a Introduction These influences modulate both the termination codon are subject to degra- levels and localization of expressed dation by the nonsense-mediated decay A clearly appreciated role for introns in proteins. While 90% of human introns (NMD) pathway [4–8]. Reflecting the higher organisms is to allow for alterna- occur within protein coding regions widespread view that NMD is restricted tive splicing, which permits a single (open reading frames; ORFs), many also to mRNAs encoding inappropriately gene to encode many different proteins. reside in untranslated regions (UTRs). truncated proteins, NCBI’s RefSeq data- Less widely appreciated, however, is Approximately 35% of human 50-UTRs base routinely excludes most 3UI-con- that the presence of an intron and the [3], and between 6% (NCBI’s taining transcripts from its annotated act of its removal by the spliceosome can Reference Sequence; RefSeq) and 16% coding transcripts [9]. Nonetheless, influence almost every step in gene (Vertebrate Genome Association; Vega) recent evidence clearly indicates that expression from transcription and poly- of human 30-UTRs are annotated as har- 5UIs and 3UIs do have important and adenylation to mRNA export, localiz- boring introns. Yet despite their preva- unique roles in the regulation of gene ation, translation, and decay [1, 2]. lence, introns in UTRs are rarely expression that should not be over- looked. Below, we describe evidence that the presence or absence of a 5UI DOI 10.1002/bies.201200073 has significant consequences for both mRNA nuclear export and cytoplasmic 1) Biochemistry and Molecular Pharmacology, Abbreviations: mRNA metabolism, and that 3UIs have University of Massachusetts Medical School, EJC, exon junction complex; mRNP, mRNA multiple roles in modulating normal Worcester, MA, USA protein particle; NMD, nonsense-mediated 2) Donnelly Centre for Cellular and Biomolecular decay; ORF, open reading frame; TREX, protein expression. Research, University of Toronto, Toronto, transcription export; UTR, untranslated region; Ontario, Canada 3UI, 30-UTR intron; 5UI, 50-UTR intron. 3) Howard Hughes Medical Institute, Worcester, Splicing directs mRNP MA, USA y These authors contributed equally to this work. formation *Corresponding author: Melissa J. Moore All introns can influence gene expres- E-mail: [email protected] sion regardless of their position relative Bioessays 34: 1025–1034,ß 2012 WILEY Periodicals, Inc. www.bioessays-journal.com 1025 A. A. Bicknell et al. Insights & Perspectives ..... to the coding region because they alter roles in exon definition and as alterna- 5UIs are present, they are necessarily the protein makeup of the mRNA tive splicing regulators. However, they the most 50 proximal introns in a tran- protein particle (mRNP). One set of are also subject to splicing-dependent script, and 50 proximal introns have a splicing-dependent mRNP proteins is dephosphorylation, which promotes disproportionate role in regulating tran- the exon junction complex (EJC), depos- their tight association with the spliced scription, mRNA export, and translation ited by the spliceosome 24 nts mRNA. As mRNP components, SR [16, 25–28]. However, 50 proximity alone upstream of exon junctions on spliced proteins can enhance nucleocytoplas- cannot explain the functional distri- mammalian mRNAs [10, 11]. This multi- mic export, translation, and decay of bution of transcripts that do or do not protein complex remains tightly bound their bound mRNAs [20–22]. Thus, as contain 5UIs. Importantly, transcripts to the mRNA until the first round of elaborated below, one means by which possessing only coding-region introns, Think again translation, when EJCs within the cod- 5UIs and 3UIs influence gene expression and in which the first intron has the ing region are displaced by ribosomes as is by promoting the loading of mRNP same proximity to the transcription start they translocate across the message proteins with downstream functional site as a 5UI, do not display the same (Fig. 1) [12]. Until then, the EJC core consequences. functional distribution as 5UI-contain- serves both as a molecular marker of ing transcripts [3]. prior intron position, and as a binding 50-UTR introns and an The enrichment of 5UIs in regulatory platform for peripheral proteins. These genes could reflect their tendency to peripheral factors associate transiently alternative mRNA have more transcription factor binding with the core and help regulate the sub- export pathway sites, which are often located within the cellular localization, translation, and first intron [26]. In addition, deposition decay of the transcript [2, 13–15]. Initial models suggested that 5UIs of splicing-dependent mRNP com- Also deposited on mRNAs during evolved under nearly neutral genetic ponents as close as possible to the 50 transcription and splicing are the tran- selection, implying that they have no end of the mRNA could play a positive scription export (TREX) complex and SR specific function [23]. If this were the role in facilitating rapid export and proteins. In mammals, the TREX com- case, one would expect 5UIs to be translation of the newly made mRNAs plex is recruited primarily to the 50 end equally distributed among transcripts [28]. of transcripts through cooperative of all functional classes. Recent On the other hand, some transcripts action of the nuclear cap-binding com- analyses, however, have revealed that have evolved to exclude introns from plex and the spliceosome [16, 17]. Once genes having or lacking 5UIs fall into their 50-UTRs because this allows them bound to the mRNA, the TREX complex distinct functional classes, at least in the to use an alternate mode of nuclear promotes nuclear export of fully proc- human and rat genomes. Whereas genes export, the ALREX mRNA export path- essed transcripts through the nuclear with regulatory functions are enriched way. Unlike the canonical TREX- pore by direct interactions between for 5UIs, genes encoding proteins tar- dependent nuclear export pathway, the TREX component, Aly, and the geted to the endoplasmic reticulum the ALREX pathway does not require nuclear export factor, TAP-p15 [18, 19]. (ER) or mitochondria are significantly splicing [29]. Instead, ALREX facilitates SR proteins are best known for their depleted of such introns [3, 24]. When mRNA export via a specific RNA sequence element located within the 50 end of the ORF [29, 30]. This sequence element is particularly prominent in transcripts encoding ER and mitochon- drial-targeted proteins, the same func- tional class that is depleted of 5UIs. The current model is that when ALREX elements are present, their position relative to the first intron dictates the method of mRNA export. For transcripts lacking a 5UI, if an ALREX sequence is present at the 50 end of the ORF, it is likely to be upstream of the first intron (which would be in the ORF). Thus, the ALREX pathway is used to export the mRNA from the nucleus. On the other hand, for transcripts containing a 5UI, the first intron is necessarily upstream of the ORF. These mRNAs are exported by the canonical TREX pathway regard- less of whether an ALREX element is Figure 1. During splicing the EJC is deposited just upstream of splice junctions. Following translation termination, nonsense-mediated decay degrades transcripts harboring an EJC present in the ORF (Fig. 2). In support >55 nts downstream of a termination codon. Degradation occurs as a result of interactions of this model, nucleotide sequences between the terminating ribosome, Upf1, Upf2, Upf3, and the EJC. near the 50 end of the ORF strongly 1026 Bioessays 34: 1025–1034,ß 2012 WILEY Periodicals, Inc. .....Insights & Perspectives A. A. Bicknell et al. Think again Figure 2. For mRNAs containing an ALREX ing between the two export pathways, and stimulate NMD when the transcript sequence, position of this sequence relative thereby regulating subsequent mRNA has a particularly long 30-UTR [38]. This to the first intron may determine route of expression. Expressed sequence tags additional form of NMD is presumably mRNA export. A: If a 5UI is present, the first (ESTs), cap analysis of gene expression splicing-independent, and is discussed intron is