Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms

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Molecular and Cellular Neuroscience xxx (2013) xxx–xxx

YMCNE-02793; No of Pages 9

Contents lists available at SciVerse ScienceDirect

Molecular and Cellular Neuroscience

j ourna l homepage: www.e lsev ie r .com/ locate /ymcne

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Regulation of gene expression in mammalian nervous system through alternativepre-mRNA splicing coupled with RNA quality control mechanisms

Karen Yap, Eugene V. Makeyev ⁎School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore
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⁎ Corresponding author at: School of Biological ScieUniversity, 60 Nanyang Drive, SBS-02n-45, Singapore

E-mail address: [email protected] (E.V. Makeyev

1044-7431/$ – see front matter © 2013 Published by Elhttp://dx.doi.org/10.1016/j.mcn.2013.01.003

Please cite this article as: Yap, K., Makeyev,splicing coupled with RNA..., Mol. Cell. Neuro

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Article history:Received 26 October 2012Accepted 17 January 2013Available online xxxx

Keywords:Post-transcriptional regulation of geneexpressionAlternative pre-mRNA splicingRNA quality controlNonsense-mediated decayNuclear retention and elimination ofincompletely spliced transcriptsNervous system development and function

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REukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mech-anisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stabilitythrough downstreamRNA quality control (QC) pathways including nonsense-mediated decay (NMD) ofmRNAscontaining premature termination codons and nuclear retention and elimination (NRE) of intron-containingtranscripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing bodyof evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns arealso used in a number of biological contexts for deterministic control of gene expression. Here we review recentstudies elucidating molecular mechanisms and biological significance of these gene regulation strategies with aspecific focus on their roles in nervous system development and physiology. This article is part of a Special Issueentitled ‘RNA and splicing regulation in neurodegeneration’.

© 2013 Published by Elsevier Inc.

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RREIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Alternative splicing coupled with nonsense-mediated decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Alternative splicing coupled with nuclear retention and elimination of intron-containing transcripts . . . . . . . . . . . . . . . . . . . . . . . . . 0Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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Introduction

The discovery that different metazoan organisms depend on large-ly similar repertoires of protein-encoding genes has suggested thatevolutionary processes within this clade may rely on elaboration oftranscriptional and posttranscriptional gene regulation mechanisms(Keren et al., 2010; Lenhard et al., 2012; Levine and Tjian, 2003;Licatalosi and Darnell, 2010; Moore and Proudfoot, 2009; Nilsen andGraveley, 2010). One such mechanism is based on alternative splicingof multiexon pre-mRNA transcripts into two or more distinct mRNAproducts (Black, 2003; Calarco et al., 2011; Nilsen and Graveley,2010; Wang and Burge, 2008).

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E.V., Regulation of gene exprsci. (2013), http://dx.doi.org

Several common alternative splicing patterns have been describedincluding cassette exons, mutually exclusive exons, alternative 5′ and3′ splice sites, alternative 5′ and 3′ exons, and alternative intron reten-tion events (Black, 2003; Wang and Burge, 2008) (Fig. 1). In each case,the choice between the alternatives is regulated through an interplay be-tween constitutive splicing motifs (5′ splice sites, branch points,polypyrimidine tracts and 3′ splice sites) and components of the coresplicing machinery, as well as optional cis-regulatory elements (exonicand intronic splicing enhancers and silencers referred to as ESE, ISE,ESS, and ISS, respectively) and a range of trans-factors (normally RNA-binding proteins, or RBPs) interacting with these elements (Black,2003; Wang and Burge, 2008).

Recent transcriptome-wide analyses suggest that >90% of humangenes may give rise to alternatively spliced transcripts (Calarco et al.,2011; Chen and Manley, 2009; Pan et al., 2008; Wang et al., 2008). Ap-proximately 100,000 intermediate- to high-abundance alternativelyspliced events have been identified with the largest fraction occurring in

ession in mammalian nervous system through alternative pre-mRNA/10.1016/j.mcn.2013.01.003

http://dx.doi.org/10.1016/j.mcn.2013.01.003

mailto:[email protected]


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Fig. 1. Possible alternative splicing topologies.Modified from Black (2003).

2 K. Yap, E.V. Makeyev / Molecular and Cellular Neuroscience xxx (2013) xxx–xxx

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the nervous system (NS) (Calarco et al., 2011; Li et al., 2007; Pan et al.,2008). Since the human genome is estimated to contain ~20,000–25,000 protein-coding genes (International Human Genome SequencingConsortium, 2004), this potentially translates to a >4-fold gain in the ef-fective coding capacity. In several exceptional cases, individual genes areknown to give rise to hundreds and sometimes thousands of distinctsplice forms (Hattori et al., 2009; Nilsen and Graveley, 2010; Park andGraveley, 2007).

Biogenesis of alternativemRNA isoformsmay additionallymodulatemRNA stability, translational efficiency and intracellular localization(Andreassi and Riccio, 2009; Barrett et al., 2012; Lareau et al., 2007a;Lutz and Moreira, 2011). Notably, 20–35% of alternatively splicedmRNAs are predicted to contain premature termination codons (PTCs)in the mRNA open reading frame (ORF) (Baek and Green, 2005; Greenet al., 2003; Lewis et al., 2003). This is expected to destabilize these tran-scripts through nonsense-mediated decay (NMD), an evolutionarilyconserved cytoplasmic mRNA quality control (QC) mechanism (Changet al., 2007; Isken and Maquat, 2007; Lareau et al., 2007a). Althoughmost of the PTC-containing transcripts likely appear due to randomsplicing errors, at least 10–20% of these may correspond to bona fidegene regulation events (Pan et al., 2006). Moreover, a subset ofunproductively spliced transcripts retaining intronic sequences maybe intercepted by a distinct RNA QC pathway that we refer to as nuclearretention and elimination (NRE) of intron-containing transcripts (Yapet al., 2012). In addition to its well-known role in quality control, NREcan be integrated into gene regulation networks (Yap et al., 2012).

Compared to other human organs, brain is known to express thelargest number of distinct alternatively spliced transcripts, which bothdiversifies the proteome and increases the gene regulation complexity(Calarco et al., 2011; Li et al., 2007; Norris and Calarco, 2012; Pan etal., 2008; Wang et al., 2008) (and see below). Here we summarize re-cent studies examining the functional interface between alternativesplicing and RNA QC in the mammalian NS. Other biological functionsof alternative splicing and RNA QC as well as the underlying molecularmechanisms have been discussed earlier in several excellent reviews(Bicknell et al., 2012; Black, 2003; Calarco et al., 2011; Chang et al.,2007; Doma and Parker, 2007; Huang and Wilkinson, 2012; Hwangand Maquat, 2011; Isken and Maquat, 2007; Kervestin and Jacobson,2012; Li et al., 2007; Nicholson et al., 2010; Schoenberg and Maquat,2012; Wang and Burge, 2008).

Alternative splicing coupled with nonsense-mediated decay

Nonsense-mediated decay has been originally described as a cyto-plasmic mRNA QC mechanism targeting aberrant transcripts thatemerge as a result of nonsense mutations and RNA-processing errors(Chang et al., 2007; Isken and Maquat, 2007; Kervestin and Jacobson,2012). Several NMD components identified by genetic screens in bud-ding yeast Saccharomyces cerevisiae and nematode Caenorhabditiselegans are evolutionarily conserved. These include the RNA-helicaseUPF1, its associated proteins UPF2 and UPF3, protein kinase Smg1 acti-vating UPF1 by phosphorylation, as well as Smg5, Smg6 and Smg7 pro-teins that interact with phosphorylated UPF1 and destabilizePTC-containing mRNAs (Chang et al., 2007; Isken and Maquat, 2007;Kervestin and Jacobson, 2012; Neu-Yilik and Kulozik, 2008; NicholsonandMuhlemann, 2010; Nicholson et al., 2010; Schoenberg andMaquat,

Please cite this article as: Yap, K., Makeyev, E.V., Regulation of gene exprsplicing coupled with RNA..., Mol. Cell. Neurosci. (2013), http://dx.doi.org

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2012). Yet two additional proteins called Smg8 and Smg9 control Smg1activity (Schoenberg and Maquat, 2012; Yamashita et al., 2009). NMDstrictly depends on mRNA translation and requires nuclear cap-bindingprotein complex CBP80/20, translation termination factors eRF1 andeRF3, cytoplasmic poly(A)-binding protein PABPC1 and a subset of cyto-plasmic mRNA degradation enzymes (Chang et al., 2007; Isken andMaquat, 2007; Kervestin and Jacobson, 2012; Nicholson et al., 2010;Schoenberg and Maquat, 2012).

In mammalian cells, PTC-containing transcripts are normally recog-nized based on the position of the translation termination codon rela-tive to the last (3′-proximal) exon–exon junction (Chang et al., 2007;Isken and Maquat, 2007; Kervestin and Jacobson, 2012). Terminationcodons located>50 nucleotides (nt) upstream of the last exon–exonjunction are typically recognized as premature, whereas stop codonslocatedb50 nt upstream or downstream are, as a rule, considered nor-mal. This rule is implemented through the deposition of so-called exonjunction complexes (EJCs)~20–25 nucleotides upstream ofmost exon–exon junctions (Le Hir et al., 2000; Sauliere et al., 2012; Singh et al.,2012). EJC is composed of 4 core subunits, eIF4AIII, MAGOH, MNL51/BTZ and Y14, and a number of associated factors including UPF3, UPF2and UPF1 (Chang et al., 2007; Isken and Maquat, 2007; Kervestin andJacobson, 2012). EJCs survive mRNA export from the nucleus to the cy-toplasm. However, they are dislodged by translating ribosomes duringthe “pioneer” round of translation unless associated with exon–exonjunctions positioned>50 nt downstream of the termination codon.mRNAs retaining one or several EJCs following the pioneer round oftranslation are normally subjected to NMD (Chang et al., 2007; Iskenand Maquat, 2007; Kervestin and Jacobson, 2012). Interestingly, re-quirements for specific NMD factors may differ for different targetsand some mammalian mRNAs containing long 3′UTRs may undergoNMD in an EJC-independent manner (Chang et al., 2007; Huang andWilkinson, 2012; Isken and Maquat, 2007; Nicholson et al., 2010).

Besides its role in eliminating aberrant mRNAs, NMD is known tocontribute to gene regulation programs through an alternativesplicing-dependent mechanism referred to as AS-NMD or sometimesRUST (from “regulation by unproductive splicing and translation”)(Lareau et al., 2007a; McGlincy and Smith, 2008; Neu-Yilik and Kulozik,2008). Different estimates predict that AS-NMDmay control the abun-dance of ~2% to 35% of alternatively spliced transcripts with a consider-able fraction of regulated splicing events showing interspeciesconservation (Baek and Green, 2005; de Lima Morais and Harrison,2010; Green et al., 2003; Lewis et al., 2003; Mudge et al., 2011; Pan etal., 2006; Zhang et al., 2009). Notably, a number of genes known to beunder AS-NMD control encode RBPs and other proteins involved in cel-lular RNA metabolism and in many of these cases, the correspondingRBPs auto-regulate the AS-NMD process through a negative feedbackloop (Cuccurese et al., 2005; Lareau et al., 2007a, 2007b; McGlincy andSmith, 2008; Ni et al., 2007; Saltzman et al., 2008, 2011).

RBPs that normally function as splicing repressors (including a largefraction of hnRNP proteins) tend to stimulate NMD of their ownmRNAsby inhibiting “ORF-maintaining” cassette exons [(McGlincy and Smith,2008) and see below] (Fig. 2A). Since the lengths of these exons arenot divisible by 3, their skipping is expected to shift the ORF registerand result in the appearance of a downstream PTC (Magen and Ast,2005). On the other hand, splicing activators (e.g., SR and hnRNP pro-teins interacting with splicing enhancer sequences) usually stimulate



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the inclusion of specialized “poison” exons that either contain in-framenonsense codons or shift the frame and expose a downstream PTC(Lareau et al., 2007a, 2007b; McGlincy and Smith, 2008; Ni et al.,2007; Rossbach et al., 2009; Saltzman et al., 2008, 2011) (Fig. 2B). Insome cases, poison exons can be mutually exclusive with coding cas-sette exons [(Lareau et al., 2007a); and see below] (Fig. 2C). Anothercommon AS-NMD strategy relies on activation of splicing of alternative3′-terminal introns located>50 nt downstream of the normal stopcodon (Lareau et al., 2007a; McGlincy et al., 2010; Sun et al., 2010;Sureau et al., 2001) (Fig. 2D). This triggers NMD since the newly assem-bled 3′-terminal EJCs cannot be accessed by translating ribosomes inthis case. It is currently unknown whether other PTC-inducing splicingevents (Lareau et al., 2007a) are involved in the AS-NMD pathway.

Recent studies have begun uncovering more sophisticated networksthat often comprise an RBP-based “regulatory hub” and one or more“subordinate” genes cross-regulated by the RBP using AS-NMD mecha-nisms. Interestingly, this paradigm appears to be extensively used inthe mammalian NS. One pertinent example involves a polypyrimidinetract-binding protein called PTBP1 (alternative names PTB and hnRNPI) and its paralog PTBP2 (also called nPTB or brPTB). In the NS-specificcontext, PTBP1 is expressed in neural stem cells (NSCs) and non-neuronal cells (e.g., glia), whereas PTBP2 is expressed predominantly inpost-mitotic neurons (Keppetipola et al., 2012). Besides their otherroles in cellular RNA metabolism, both PTBPs function as global regula-tors of the neuron-specific alternative splicing program (Boutz et al.,

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Fig. 2. Four common strategies of AS-NMD. The gene structures are presented in the middbottom of each diagram. Splicing scenarios leading to normal expression are shown at the


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2007; Makeyev et al., 2007; Spellman et al., 2007). PTBP1 is essentialfor early mouse embryogenesis (Shibayama et al., 2009; Suckale et al.,2011) and a NS-specific deletion of this gene leads to a loss of adherensjunctions in the ventricular zone of the dorsal telencephalon, prematureneuronal differentiation, defects in gliogenesis, and ultimately, lethal hy-drocephalus (Shibasaki et al., 2012). Similarly,mice lacking PTBP2 exhibitneuronal progenitor defects and premature neurogenesis and die soonafter birth (Licatalosi et al., 2012).

Notably, PTBP1 auto-regulates its expression through a homeostaticAS-NMD mechanism (Wollerton et al., 2004). When present at highlevels, PTBP1 protein binds to its own pre-mRNA in the vicinity of a34 nt-long cassette exon (exon 11) and stimulates its skipping. Thisleads to a frame shift exposing a PTC in exon 12 and ultimately culmi-nates in degradation of the PTBP1 mRNA (Wollerton et al., 2004). A de-crease in the intracellular PTBP1 protein concentration promotes exon11 inclusion thus giving rise to relatively stable and translationally ac-tive mRNA species (Wollerton et al., 2004). Despite this homeostaticregulation, PTBP1 levels are markedly reduced in developing neurons,at least in part due to binding of a neuron-enriched microRNA miR-124 to the PTBP1 mRNA 3′UTR (Makeyev et al., 2007). This triggers aglobal shift towards brain-specific alternative pre-mRNA splicing pat-terns (Boutz et al., 2007; Llorian et al., 2010; Makeyev et al., 2007;Spellman et al., 2007; Xue et al., 2009).

One of these splicing changes affects the ORF-maintaining cassetteexon 10 in the PTBP2 pre-mRNA and enables AS-NMD regulation of

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le and the corresponding alternative regulation outcomes are depicted at the top andtop and those inducing NMD are shown at the bottom.



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the PTBP2 expression levels (Boutz et al., 2007; Makeyev et al., 2007;Spellman et al., 2007). In non-neuronal cells, PTBP1 protein repressesthis exon and leads to the production of PTC-containing PTBP2 mRNAspecies,which are degraded byNMD.A decrease in the PTBP1 expressionduring neurogenesis stimulates the inclusion of this exon thus aug-menting the production of functional PTBP2 mRNA and ultimatelyPTBP2 protein in newly born neurons (Boutz et al., 2007; Makeyev etal., 2007; Spellman et al., 2007) (Fig. 3A). The exon 10 inclusion is addi-tionally stimulated by the NS-specific SR protein nSR100/SRRM4(Calarco et al., 2009). PTBP1 and PTBP2 are known to control overlappingsets of alternatively spliced elementswith PTBP1 being generally a stron-ger regulator (Keppetipola et al., 2012; Licatalosi et al., 2012; Llorian etal., 2010; Makeyev et al., 2007; Xue et al., 2009; Zheng et al., 2012). Asneuronsmature, thePTBP2protein levels gradually decrease thus furtherenforcing neuron-specific alternative splicing patterns (Licatalosi et al.,2012; Zheng et al., 2012). Although themechanismunderlying this effectis presently unknown, the 3′UTR of the PTBP2mRNA contains functionalmiR-124 binding sites (Makeyev et al., 2007), which might potentiallydiminish the translational efficiency or/and stability of thismRNA inma-ture neurons.

At least two additional NS-specific genes are known to be regulatedby PTBP1 in an AS-NMD-dependent manner. One of these genes,Gabbr1, encodesmetabotropic GABAB1 receptor localized predominant-ly (but not exclusively) at postsynaptic terminals of inhibitory synapses(Pinard et al., 2010). Repression of the Gabbr1 cassette exon 15 by

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Fig. 3. (A). Extended AS-NMD circuitry controlled by PTBP1 in the NS. (B) AS-NMD circuitry reduring neuronal differentiation are indicated bywedges on the left and corresponding regulatioresponding neuronal proteins is indicated by thin dashed lines.


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PTBP1 shifts the frame and transforms Gabbr1 mRNA into an NMD tar-get. A decrease in the PTBP1 protein levels in neurons undergoing dif-ferentiation stimulates exon 15 inclusion and rescues the Gabbr1mRNA abundance (Makeyev et al., 2007). The other gene,Dlg4, encodesan abundant postsynaptic protein PSD-95 regulating structure andfunction of excitatory synapses (Sheng and Hoogenraad, 2007). Micelacking PSD-95 exhibit spatial learning defects accompanied by en-hanced glutamate receptor-dependent long-term potentiation (LTP)and reduced long-term depression (LTD) (Migaud et al., 1998). TheAS-NMDmechanism underlying Dlg4/PSD-95 regulation is conceptual-ly similar to those described above for PTBP2 and Gabbr1; however, theORF-maintaining cassette exon 18 of Dlg4/PSD-95 is not fullyde-repressed until later stages of neuronal differentiation when bothPTBP1 and PTBP2 levels are diminished (Zheng et al., 2012) (Fig. 3A).Thus, sequential down-regulation of PTBP1 and PTBP2 ensures a gradu-al accumulation of PSD-95 during brain development.

The mammalian Rbfox family comprising Rbfox1 (A2BP1/Fox-1),Rbfox2 (RBM9/Fox-2) and Rbfox3 (HRNBP3/Fox-3/NeuN) providesanother example of an extended AS-NMD circuitry. Similar toPTBP1 and PTBP2, these RBPs are involved in the regulation of a num-ber of important NS-specific alternative splicing events (Gehman etal., 2011, 2012; Underwood et al., 2005). In the NS, Rbfox1 andRbfox3 are expressed in postmitotic brain regions whereas Rbfox2shows a more ubiquitous expression pattern (Gehman et al., 2011,2012; Kim et al., 2009; McKee et al., 2005; Underwood et al.,

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gulated by Rbfox proteins. (A–B) Changes in the PTBP1 and Rbfox protein concentrationsn outcomes are shown at the top and bottom of each diagram. Cellular localization of cor-



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2005). Knockout mice lacking Rbfox1 in the NS develop an increasedsusceptibility to spontaneous and kainic acid-induced seizures, whereasNS-specific expression of Rbfox2 is essential for proper developmentand mature physiology of the cerebellum (Gehman et al., 2011, 2012).

The expression of Rbfox2 in the NS appears to be limited through al-ternative splicing of mutually exclusive coding cassette exon 6 and twoadjacent poison exons 5* and 6* (Dredge and Jensen, 2011). Rbfox1 andRbfox3 proteins interacting with conserved binding sequences adjoin-ing exon 6 repress the inclusion of this exon and give rise to a dominantnegative (FoxΔRRM) Rbfox2 protein (Damianov and Black, 2010).However, a further analysis of this system shows that the repressionof exon 6 by Rbfox3 (and potentially by Rbfox1) may additionally stim-ulate the utilization of poison exons 5* and 6* thus committing a frac-tion of the Rbfox2 transcripts to the NMD pathway (Dredge andJensen, 2011).

Notably, conditional inactivation of both Rbfox1 and Rbfox2 genes inPurkinje neurons results in highly irregular firing patterns due to re-duced expression of the NS-specific voltage-gated sodium channelScn8a/Nav1.6 (Gehman et al., 2012). The Scn8a pre-mRNA containstwo mutually exclusive exons enabling AS-NMD regulation: poisonexon 18N utilized at embryonic and neonatal stages and coding cassetteexon 18A activated in the adult brain (Gehman et al., 2012; O'Brien etal., 2012; Zubovic et al., 2012). Importantly, the adult mouse neuronslacking Rbfox1 and Rbfox2 fail to complete the switch from 18N to 18A(Gehman et al., 2012). Moreover, interaction of any of the three Rbfoxproteins with a downstream intronic enhancer stimulated the inclusionof exon 18A in vitro (O'Brien et al., 2012; Zubovic et al., 2012). Thus, ex-pression of appropriate levels of all three Rbfox proteins in adult brain islikely required to change the AS pattern of the Scn8a mRNA and rescueit from NMD.

Other AS-NMD circuitries operating in the NS context include thoseregulated by SRSF3/SRp20 and hnRNP L proteins. Of these, SRSF3auto-regulates its own homeostasis and uses AS-NMD mechanisms tocross-regulate the expression of other SR proteins including SRSF2/SC35, SRSF5/SRp40 and SRSF7/9G8 (Anko et al., 2012). Since SRSF3levels are known to increase during neuronal differentiation (Anko etal., 2010), this mechanism is likely to alter the NS-specific SR proteinrepertoire. hnRNP L relies on a poison exon strategy to both auto-regulate its own expression and cross-regulate the abundance of itsparalog hnRNP LL (Rossbach et al., 2009). Although hnRNP L is a ubiqui-tously expressed protein, it contributes to activity-dependent alterna-tive splicing regulation of important neuronal genes (Liu et al., 2012;Yu et al., 2009).

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Fig. 4. (A) General outline of the AS-NRE pathway. The gene structure is shown in themiddle oretention scenario promoting NRE is at the bottom. (B) Summary of AS-NRE-mediated regulat


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The general significance of the NMD pathway for NS developmentand function has been underscored by the discovery that the core EJCcomponent eIF4AIII associates with translationally silent neuronalmRNA granules and modulates the expression of Arc/Arg3.1, a naturalNMD target contributing to multiple forms of synaptic plasticity (Giorgiet al., 2007). This is in line with the defects in synaptic architecture andsynaptic strength observed in Drosophila Smg1 mutants (Long et al.,2010) and the mental retardation symptoms in patients containing mu-tations in the UPF3X/UPF3B gene encoding one of the two human UPF3paralogs (Tarpey et al., 2007). Interestingly, the efficiency of NMD maybe naturally attenuated during normal NS development due to increasedexpression ofmicroRNAmiR-128 targeting UPF1 and the EJC componentMLN51 (Bruno et al., 2011). This mechanismmay lead to at least partialde-repression of hundreds of constitutively spliced NS-specific tran-scripts containing NMD-promoting features at appropriate developmen-tal stages (Bicknell et al., 2012; Bruno et al., 2011; Giorgi et al., 2007;Karam and Wilkinson, 2012).

Alternative splicing coupledwith nuclear retention and eliminationof intron-containing transcripts

Several RNA QC mechanisms have been identified in the nucleus(Doma and Parker, 2007; Egecioglu and Chanfreau, 2011; Fasken andCorbett, 2009; Schmid and Jensen, 2010; Sommer and Nehrbass,2005). In one specific example that we refer to as NRE (nuclear reten-tion and elimination of intron-containing transcripts), incompletelyspliced transcripts are retained in the nucleus and – should theunspliced introns fail to be removed within biologically meaningfultimeframe – eventually cleared by nuclear RNA degradation enzymes(Chang and Sharp, 1989; Doma and Parker, 2007; Houseley andTollervey, 2009; Legrain and Rosbash, 1989; Lemieux et al., 2011;Schmid and Jensen, 2010) (Fig. 4A).

Although the molecular details of NRE have not been understoodcompletely, this mechanism is known to require interaction betweencomponents of the core splicing machinery with the intronic 5′ and the3′ splice sites (Kaida et al., 2007; Rain and Legrain, 1997; Takemura etal., 2011). Incompletely spliced transcripts may be retained in the nucle-ar speckles (Girard et al., 2012; Johnson et al., 2000; Kaida et al., 2007) or/and at the nucleoplasmic face of the nuclear pore complex (Coyle et al.,2011; Galy et al., 2004; Palancade et al., 2005; Rajanala and Nandicoori,2012; Sommer and Nehrbass, 2005; Yap et al., 2012). Some intron-containing viral and cellular transcripts may contain specialized cis-elements recruiting RNA export factors that override nuclear retention

f the diagram; complete splicing scenario leading to normal expression is at the top; intronion of presynaptic genes by PTBP1. See text for details.



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and enable translocation of incompletely spliced RNAs to the cytoplasm(Cullen, 2003; Galante et al., 2004; Li et al., 2006). Conversely, othercis-elements may increase the efficiency of RNA retention in the nucleus(Taniguchi et al., 2007).

Similar to AS-NMD, NRE coupled with intron-specific alternativesplicing (AS-NRE) has been recently shown to function as a determinis-tic gene regulation strategy. For example, regulated intron retention isknown to coordinate expression of meiotic and ribosomal proteingenes in budding and fission yeasts (Averbeck et al., 2005; Cremona etal., 2011; Lemieux et al., 2011; Moldon et al., 2008; Munding et al.,2010; Parenteau et al., 2011). Similarly, human SR protein SRSF1/ASF/SF2 has been recently shown to homeostatically control its own expres-sion by promoting accumulation of incompletely spliced SRSF1 mRNAspecies in the nucleus, in addition to other auto-regulatorymechanisms(Sun et al., 2010).

Several recent studies have suggested that AS-NRE may contributeto mammalian NS development and function. One relevant example isprovided by apolipoprotein E (ApoE), a stress response protein thathas been linked with etiology of Alzheimer's and some other neurode-generative diseases (Adibhatla and Hatcher, 2008). ApoEmRNA retainsintron 3 under normal conditions, which arrests themRNA export fromthe nucleus to the cytoplasm and limits the ApoE protein productionoutput (Xu et al., 2008). However, excitotoxic stress stimulates splicingof this exon through a poorly understoodmechanism, thus allowing ac-cumulation of translation-competent ApoEmRNA in the cytoplasm (Xuet al., 2008).

Another recently identified AS-NRE circuitry regulates the expres-sion of several presynaptic proteins including Stx1b (tSNARE), Vamp2(vSNARE), Sv2a (synaptic vesicle marker) and Kif5a (neuron-specifickinesin subunit) (Yap et al., 2012). Surprisingly, these neuron-specificgenes are transcribed at detectable levels in both neurons and non-neuronal cells. However, splicing of their 3′-terminal introns is repressedin non-neuronal cells by PTBP1 protein interacting with intronicpyrimidine-rich sequences (Yap et al., 2012). This, in turn, hinders theexport of the incompletely spliced transcripts from the nucleus to the cy-toplasm and triggers their nuclear elimination. The latter appears to de-pend on the exosome complex and the nuclear pore-associated proteinTpr/Mlp1 (Yap et al., 2012). Moreover, NRE depends on a cross-talk be-tween retained introns and the splicingmachinery. Indeed, genetic abla-tion of the 5′ splice site within a PTBP1-controlled intron was shown toabolish the regulation (Yap et al., 2012). A decrease in the PTBP1 levelsduring neuronal differentiation allows the 3′-terminal introns to bespliced out thus allowing translation-competent mRNAs to accumulatein the cytoplasm. This mechanism likely safeguards non-neuronal cellsfrom ectopic and precocious expression of functionally linked presynap-tic genes and ensures their coherent activation in neurons.

Of note, TDP-43/TARDBP, an hnRNP protein mutated in some pa-tients with amyotrophic lateral sclerosis and frontotemporal dementia,might be auto-regulated through a similar, albeit not identical mecha-nism (Avendano-Vazquez et al., 2012; Ayala et al., 2011; Sreedharan etal., 2008). In this case, an increase in the TDP-43 expression levels pro-motes recruitment of the TDP-43 protein to the TDPBR sequence withinits own pre-mRNA, which changes the cleavage/polyadenylation sitepreference from the proximal pA1 to the distal pA2 (Avendano-Vazquez et al., 2012). The pA2-terminated transcripts are retained anddegraded in the nucleus providing a mechanism for maintaining theTDP-43 homeostasis. Interestingly, interaction of the TDP-43 proteinwith TDPBR also stimulates intronic definition of a large 3′-terminal seg-ment comprising the pA1 site (intron 7) (Avendano-Vazquez et al.,2012). Although this intron appears to be spliced out, the efficiencyand the time course of this reaction have not been investigated. It istherefore formally possible that, prior to splicing, intron 7 may committhe TDP-43 mRNA to NRE. This would be generally consistent with thedependence of the TDP-43 auto-regulation on the intron 7 functionality(Avendano-Vazquez et al., 2012), evocative of the PTBP1-dependent reg-ulation of presynaptic proteins (Yap et al., 2012).


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Conclusions and future directions

The growing list of genes known to be controlled by AS-NMD andAS-NRE argues for functional utility and evolutionary success of theseregulation strategies. This may seem surprising since both mechanismsinvolve thermodynamically futile cycles of RNA transcription and deg-radation. We envision several situations where advantages of AS-NMDand AS-NRE likely outweigh this limitation. First, specialized AS-NMDand AS-NRE circuitries facilitating auto- and cross-regulation of RBPsand other proteins involved in RNAmetabolismmay provide an exam-ple of using parsimonious design principles to increase robustness of bi-ological networks (Leclerc, 2008). Indeed, harnessing RNA-bindingactivity of an RBP in this context bypasses the need for additional regu-lators and streamlines the network architecture. Second, extendedAS-NMD and AS-NRE circuitries comprising an RBP “hub” and at leastone “subordinate” gene (e.g., the network depicted in Fig. 3B)may buff-er the expression noise of the “subordinate” geneby linking it to the RBPexpression levels, which are normally stabilized by auto-regulatoryfeedback loops. This feature may enhance the robustness of cellulargene expression programs. Third, placing several functionally linked“subordinate” genes under an RBP control (e.g. Figs. 3A and 4B)may ad-ditionally ensure their co-regulation. In a specific example of this sce-nario, AS-NMD and AS-NRE might be used to dampen the levels of“leaky” transcripts of neuronal genes inappropriately expressed innon-neuronal cells containing large amounts of PTBP1 (Makeyev etal., 2007; Yap et al., 2012; Zheng et al., 2012). Finally, several post-transcriptional strategies have been implicated in coordinating the ex-pression of functionally linked gene sets (Keene, 2007). However,AS-NMD and AS-NRE function upstream of the cytoplasmic mecha-nisms regulating mRNA translational efficiency, stability and localiza-tion, which might be critical to enable accelerated changes in geneexpression levels in response to environmental cues or during rapid de-velopmental transitions (Lareau et al., 2007a; Xu et al., 2008).

Given the exquisite structural and functional complexity of the brainand the prevalence of alternative pre-mRNA splicing in this organ, anumber of additional NS-specific AS-NMD and AS-NRE circuitries willbe likely uncovered in the future using advanced data mining and func-tional approaches (Barash et al., 2010; de Lima Morais and Harrison,2010; Ip et al., 2011; Yap et al., 2012). Of note, the 3′UTR lengths of mul-tiple mRNA species are known to be regulated in a cell-specific mannerby alternative cleavage/polyadenylation mechanisms (Berg et al., 2012;Dai et al., 2012; Ji and Tian, 2009; Mansfield and Keene, 2012; Mayrand Bartel, 2009; Sandberg et al., 2008). Since the length of the 3′UTRis known to modulate the efficiency of EJC-independent NMD (Changet al., 2007; Huang and Wilkinson, 2012; Isken and Maquat, 2007;Nicholson et al., 2010), detailed analyses of these alternatively processedtranscripts may potentially identify additional biologically importantNMD targets.

Moreover, recent RNA-seq analyses have shown that human embry-onic brain expresses a substantially larger fraction of intron-containingtranscripts than does adult brain and that this network might be con-trolled by important splicing regulators TDP-43 and NOVA (Ameur etal., 2011).Whether these intron-containing transcripts eventuallyfinal-ize their splicing and nucleocytoplasmic export to become functionalmRNAs or instead succumb to NREwill be an important question for fu-ture studies. It will also be interesting to examine possible NS-specificroles of other forms of cellular RNA QC including nonsense-mediatedtranslational repression (Lee et al., 2010; McGlincy et al., 2010; You etal., 2007), Staufen-mediated decay (Gong and Maquat, 2011; Kim etal., 2005; Maquat and Gong, 2009), non-stop decay and no-go decay(Graille and Seraphin, 2012), as well as retention of ADAR-modifiedtranscripts in the nuclear paraspeckles (Chen et al., 2008; Prasanth etal., 2005).

In conclusion, alternative splicing coupled with RNA QC appears tofunction in the NS as a prevalent gene regulation strategy. We predictthat future work in this area will yield additional important insights



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into molecular mechanisms that allow the information stored in theform of genomic DNA to be converted into brain structure and function.

Acknowledgments

We thank Snezhka Oliferenko and members of the Makeyev lab forvaluable discussions. Thisworkwas supported by theNational ResearchFoundation Singapore (grant NRF-RF2008-06, EVM). We apologize toour colleagues whose work could not be cited due to space limitations.

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Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms