Aberrant RNA processing events in neurological disorders

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available at www.sciencedirect.com

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Review

Aberrant RNA processing events in neurological disorders Karen Anthony⁎, Jean-Marc Gallo MRC Centre for Neurodegeneration Research, King's College London, Department of Clinical Neuroscience, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK

A R T I C LE I N FO

AB S T R A C T

Article history:

The importance of aberrant RNA processing in neurodegeneration is becoming increasingly

Accepted 3 March 2010

clear; a recent example being the identification of the splicing factor TDP-43 as the major

Available online 10 March 2010

component of inclusions characteristic of a number of neurodegenerative conditions including amyotrophic lateral sclerosis (ALS). Due to the enormous diversity generated by

Keywords:

alternative splicing and its importance in the nervous system, it is no surprise that defective

RNA processing

alternative splicing in disease has been particularly well documented. However, in addition

Neurodegeneration

to splicing, other RNA processing events such as RNA editing, polyadenylation and mRNA

RNA-binding protein

stability are also disrupted in some neurological disorders. For instance: the editing efficiency of specific ionotropic receptors is reduced in ALS affecting ion permeability and the function of RNA-processing proteins is affected by their sequestration to trinucleotide repeat expanded mRNAs in several disorders. Due to the extensive coupling between RNA processing events and the multifunctionality of the RNA processing factors that regulate them, it is important to consider RNA processing as a whole. Here we review RNA processing events and their extensive coupling to one another and detail the associations of RNA processing including, but not exclusively, alternative splicing with neurodegeneration. © 2010 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . Post-transcriptional mRNA processing . . . . . Neurological diseases associated with aberrant 3.1. RNA editing . . . . . . . . . . . . . . . . 3.2. Alternative splicing . . . . . . . . . . . 3.3. Polyadenylation . . . . . . . . . . . . . 3.4. Nuclear mRNA export . . . . . . . . . . 3.5. mRNA localisation and stabilisation . . 4. Concluding remarks . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, Department of Clinical Neuroscience, PO37, De Crespigny Park, London SE5 8AF, UK. Fax: +44 20 7708 0017. E-mail address: [email protected] (K. Anthony). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.03.008

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1.

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Introduction

The vast majority of neurological diseases are characterised by the presence of abnormal protein aggregates. These lesions can be in the form of extracellular protein deposits that develop as senile plaques or as intracellular inclusions within the cell bodies, nuclei and processes of neurones. As a result of these defining pathological features, neurological diseases are often thought of as diseases of protein metabolism. However, increasing evidence highlights the importance of defective RNA processing in disorders of the nervous system. For example, the RNA-binding protein TDP-43 is a major component of inclusions characteristic of a number of neurodegenerative conditions including ALS (Arai et al., 2006; Kwong et al., 2008; Neumann et al., 2006). Furthermore, mutations in the TDP-43 gene, as well as in the FUS gene, also encoding an RNA-binding protein, have been identified thus confirming their pathological importance (Kwiatkowski et al., 2009; Lagier-Tourenne and Cleveland, 2009; Sreedharan et al., 2008; Vance et al., 2009). Eukaryotic pre-messenger (m)RNA undergoes extensive post-transcriptional processing through a series of tightly coupled events such as capping, splicing and polyadenylation which protect the transcript on its journey to the cytoplasm and enhance the initiation of translation. In addition, it is thought that the complexity of the human proteome is brought about through processes such as alternative splicing and RNA editing which alter the coding capacity of a transcript. Whilst aberrant alternative splicing has been well documented in neurological disease (Gallo et al., 2005; Licatalosi and Darnell, 2006), it is evident that other RNA processing events such as RNA editing and polyadenylation are also disrupted in some disorders (AbuBaker and Rouleau, 2007; Kawahara et al., 2004). The purpose of this review is to briefly review RNA processing and detail aberrations in a variety of these intricately coupled events that are involved in neurological disease.

2.

Post-transcriptional mRNA processing

From its transcription in the nucleus to its eventual translation in the cytoplasm, RNA is accompanied by a myriad of proteins that control and regulate its fate. Eukaryotic premRNA must undergo multiple post-transcriptional processing events such as capping, splicing and polyadenylation, and although these classical steps are described sequentially, they occur co-transcriptionally in the nucleus whereby multiple mRNA processing factors are recruited to the C-terminal domain (CTD) of RNA polymerase II (Fong and Bentley, 2001). In fact it has emerged that both sequential and non-sequential coupling exists between all RNA processing events including between the first and last steps (Maniatis and Reed, 2002). The first stage in the maturation of eukaryotic pre-mRNA is the addition a “cap” to the 5′ end of the emerging nascent transcript. During this process, the 5′ terminal phosphate is removed and replaced by a guanosine 5′-monophosphate to form an unusual 5′-5′-triphosphate linkage that is protected from ribonucleases; the N-7 position of the new guanine ring is subsequently methylated to provide further mRNA protection (Shatkin, 1976). Capping is therefore achieved rapidly

during transcription when the nascent transcript is only ∼30 nucleotides long. Protein complexes assemble on this cap structure to enhance and co-ordinate downstream events such as splicing, nuclear mRNA export, stability and translation (Shatkin and Manley, 2000). Splicing is responsible for the removal of non-coding introns from a pre-mRNA and requires the accurate identification of poorly conserved cis-acting donor and acceptor sequences or splice sites at the ends of the exons. Exon definition represents a fundamental problem in pre-mRNA splicing whereby short exons must be identified within the vast stretches of intronic RNA. The coupling of splicing to transcription most likely plays an important role by positioning components of the splicing machinery in the immediate vicinity of the emerging pre-mRNA thus decreasing competition between non-specific RNAbinding proteins (Maniatis and Reed, 2002). Even so, splice sites are loosely conserved and redundant in the mammalian genome and their mutation can lead to the recognition of cryptic splice sites not normally recognised (Maniatis and Tasic, 2002). Therefore, additional information is required for exon recognition in the form of regulatory sequences that lie within exons and adjacent introns. These cis elements are recognised by several trans-acting factors that assemble into a large multicomponent ribonucleoprotein (RNP) complex, the spliceosome which carries out the splicing reaction in two transesterification reactions (Matlin and Moore, 2007). The spliceosome is a large dynamic macromolecular machine that consists of small U-rich nuclear ribonucleic acids (snRNAs) each of which is associated with several proteins to form snRNA particles, or snRNPs. In addition, non-snRNP proteins referred to as splicing factors are also recruited to the spliceosome to aid in intron removal. Spliceosome assembly is a highly ordered energy-dependent stepwise process involving the formation of distinct complexes of snRNPs and protein factors whose energy-dependent structural re-organisation brings the two splice sites closer together for intron excision (Matlin and Moore, 2007). Splicing can be achieved in an alternative fashion where two different protein isoforms can be generated from the same gene as a result of the exclusion of a particular exon from one transcript. Processes such as alternative splicing are considered responsible for human complexity rather than our surprisingly low number of genes. Genome-wide analysis of the tissue specificity of alternative splicing finds that the brain has the largest group of tissue-specific alternatively spliced isoforms (Lee and Irizarry, 2003). In the nervous system, alternative splicing coordinates the activity of protein networks at the synapse (Ule et al., 2005) and is important for genes involved in information processing such as those coding for receptors, signal transduction proteins, transcription factors and splicing regulators. Alternative splicing can modulate the functional properties of neuronal receptors by altering their affinity to agonists or changing their localisation. One such example is the NMDA R1 receptor gene that, depending on the presence or absence of the exon, C1, in the transcript, produces a protein localised to the cell surface membrane or in the cytoplasm, respectively (Lee and Irizarry, 2003). Just like the 5′ end of mRNA, the 3′ end must also be posttranscriptionally modified to be protected against ribonucleases. Polyadenylation is the addition of up to 250 adenine residues to the 3′ end to form a poly(A) tail which in humans can be more than 10,000 nt downstream of the termination site

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(Shatkin and Manley, 2000; Tian et al., 2005). Polyadenylation begins by the recognition of a polyadenylation signal sequence, the mRNA is then cleaved with the help of cleavage factors and polyadenylated by poly(A) polymerase. In contrast to the capping reaction at the 5′ end, 3′ mRNA processing requires greater complexity in the protein machinery with multisubunit factors and dozens of polypeptides required for the recognition of polyadenylation signals and polyadenylation itself (Shatkin and Manley, 2000). Akin to capping and alternative splicing, polyadenylation-associated proteins are also associated with the CTD of RNA polymerase II, thus polyadenylation most likely begins co-transcriptionally and continues post transcriptionally (Neugebauer, 2002; Shatkin and Manley, 2000). Whilst alternative splicing is by far the most common method employed to increase the coding capacity of a transcript, other RNA processing events can also contribute. For example, the use of an alternative polyadenylation site can shorten the length of the coding region or alter the length of the 3'untranslated region (3'UTR) affecting mRNA localisation, stabilisation and translation (An et al., 2008; Sandberg et al., 2008; Tian et al., 2007). Increased proteomic diversity can also be achieved by RNA editing whereby individual bases of an mRNA can be chemically altered. Nucleotides maybe changed, deleted or inserted which can affect the transcripts sequence, secondary structure, splicing, interaction with RNA-binding proteins and degradation (Agranat et al., 2008; Mattick and Mehler, 2008). The transition from adenosine to inosine (A-to-I) by adenosine deaminases acting on RNAs (ADARs) is particularly dominant, nucleotides targeted for A-toI editing reside within partially double stranded structures that are specifically recognised by ADARs (Mattick and Mehler, 2008). In the CNS, RNA editing is a highly active process responsible for the modification of transcripts that encode proteins such as ion channels involved in fast neural transmission. RNA editing is a primary means whereby environmental stimuli can alter encoded genetic information and RNA editing events have been associated with altered brain functions such as learning (Mattick and Mehler, 2008). The sites of transcription and translation are separated by the nuclear envelope; mature mRNAs made in the nucleus must be transported to the cytoplasm for translation in the form of export-competent mRNP complexes via the nuclear pore complex (NPC). The splicing of pre-mRNA is normally coupled to its nuclear export (Luo et al., 2001). In vitro, the export of spliced mRNAs is more efficient than identical mRNAs generated without splicing (Luo and Reed, 1999). Thus, a nucleoprotein complex produced as a result of splicing is most likely responsible for the efficient export of spliced mRNA. For instance, the RNA helicase UAP56 doubles as both a splicing and export factor. It is retained on spliced mRNA coupling splicing to mRNA export by directly interacting with and recruiting the export protein Aly (Luo et al., 2001). Instead of being translated immediately post nuclear export, some mRNA transcripts are targeted to specific sites such as the axons and/or dendrites of neurones for local translation. mRNA and protein localisation are important for the development and maintenance of neuronal polarity with neurones being one of the most highly polarised cells in the body. Regulatory complexes of RNA and protein control mRNA stabilisation, subcellular localisation and local translation resulting in protein segregation. The subcellular localisation of mRNA is determined by cis-elements

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(often referred to as “zipcodes”), the majority of which are located in the 3'UTR (Chabanon et al., 2004). The 3'UTR is also important for mRNA stabilisation and the regulation of translation, for example the 3'UTR harbours binding sites for non-coding microRNAs which mostly mediate negative post-transcriptional regulation (Mehler and Mattick, 2006). Zipcode sequences form secondary structures that are bound by trans-acting factors which determine the localisation and stabilisation of mRNA transcripts. Such RNA-bound trans-acting factors exist as RNP granules and have been associated with motor proteins such as kinesin that transport the targeted mRNA to sites of local protein translation along microtubules (Kanai et al., 2004; Sotelo-Silveira et al., 2006). During their transport, some mRNAs are translationally inactive; repression of translation can be at the initiation or elongation stages depending on whether the mRNA is associated with the ribosome. For example, the zipcode-binding protein 1 (ZBP1), a trans-acting factor involved in β-actin mRNA localisation to neuronal growth cones, is suggested to act simultaneously as a translational repressor (Tiedge, 2005). In neurones, the targeting of specific mRNAs to dendrites is well documented and allows rapid protein translation at postsynaptic sites in response to stimulation by neurotransmitters or neurotrophic factors. Conversely, the localisation of mRNAs to the axon has been somewhat controversial. Although the presence of RNA was first documented in the axon in the 1960's (Edström, 1966; Edstrom et al., 1962) and evidence for the presence of protein-synthesising machinery using electron microscopy was to follow (Tennyson, 1970; Yamada et al., 1971), it was largely ignored until 40 years later, when it was convincingly demonstrated that mRNAs are both present and locally translated in the axon (Piper and Holt, 2004). As a result of the discovery of axonal transport as a means of delivering proteins to the axon (Droz and Leblond, 1963) and the failure of historic studies to identify ribosomes in axons (Lasek et al., 1973), for decades, the prevailing view was that axonal proteins are synthesised exclusively in the cell body and transported to the axon via axonal transport. However, the short half lives of proteins such as tubulin raised the question as to whether proteins destined for distal parts of the axon can survive long journeys of up to a metre in length (Piper and Holt, 2004). Axonal translation is now considered to be an important aspect of synaptogenesis, long-term facilitation, memory storage, growth cone navigation and axonal regeneration (Piper and Holt, 2004; Twiss et al., 2000; Zheng et al., 2001). Given the extensive coupling between RNA processing events, it is not surprising that many of their regulatory factors are multifunctional with some having both nuclear and cytoplasmic roles (Barreau et al., 2006; Krecic and Swanson, 1999; Sanford et al., 2005). Defects in such a co-ordinated display of RNA metabolism could therefore have profound cellular implications; indeed many human diseases are caused by and associated with aberrant mRNA processing (Table 1).

3. Neurological diseases associated with aberrant RNA processing 3.1.

RNA editing

One hypothesis for the selective degeneration and death of motor neurones in sporadic ALS is a vulnerability of motor

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Table 1 – RNA processing defects associated with neurological disease. Neurological disorder RNA editing

Alternative splicing

Amyotrophic lateral sclerosis (ALS); Reduced editing efficiency of the GluR2 AMPA Alzheimer's disease (AD); schizophrenia receptor subunit at the Q/R site. and Huntington's disease (HD) Epilepsy Increased editing efficiency of the GluR2 AMPA receptor subunit at the R/G site. Altered Q/R site editing of the GluR6 kainate receptor subunit. Depression Altered RNA editing of the serotonin 5-HT2C receptor. Spinal muscular atrophy (SMA) Loss of survival for motor neurone gene 1 (SMN1) cannot be functionally compensated for by its paralog SMN2 that produces a truncated protein via enhanced exon skipping. ALS Cytoplasmic accumulation of splicing factors, TDP-43 and FUS, may interfere with the splicing of target RNAs. Abnormal expression of peripherin splice variants. Loss of astroglial glutamate transporter EAAT2 due to aberrant splicing. Frontotemporal dementia with Several FTDP-17 causing MAPT gene mutations Parkinsonism linked to chromosome affect the alternative splicing of tau exon 10. 17 (FTDP-17) Myotonic dystrophy type 1 and 2 Sequestration of splicing factors to CUG and CCUG (DM1, DM2) repeat expanded mRNA affects the splicing of target mRNAs in DM1 and DM2 respectively. Rett syndrome (RTT) Mutations in the causative gene, MECP2, affect splicing of target mRNAs. Sporadic AD Overexpression of RNA-binding protein HMGA1a alters the splicing of presenilin-2. The H1 MAPT haplotype confers increased AD susceptibility and is associated with an increase in tau exon 10-containing transcripts. A polymorphism in the promoter region of the essential splicing and export factor UAP56 is associated with a reduced risk of AD and UAP56 mRNA levels are upgregulated in AD. Lethal congenital contracture syndrome Disease-causing mutations in the GLE1 gene type 1 (LCCS1) and lethal arthrogryposis result in a predicted mis-splicing event. with anterior horn cell disease (LAAHD) Fragile X-associated tremor/ataxia Pre-mutation repeat expansions in the FMR1 syndrome (FXTAS) gene results in splicing factor sequestration. Paraneoplastic opsoclonus-myoclonus- Autoimmune response against the neurone ataxia (POMA) specific splicing factor, Nova1. Schizophrenia Upregulation of DISC1 splice variants. Spinocerebellar ataxia (SCA) types 2, 8, Splicing factors interact with several ataxia10 and 12 causing proteins. SCA1

Polyadenylation Oculopharyngeal muscular dystrophy (OPMD) Nuclear mRNA export

Comments

OPMD

X-linked mental retardation (XLMR) Fragile X syndrome (FXS)

LCCS1 and LAAHD DM1 and DM2

Enhanced interaction of ataxin-1 with splicing factor RBM17 Polyalanine expansion and aggregation of polyadenylation binding protein nuclear 1 (PABPN1). Sequestration of proteins involved in nuclear mRNA export and altered trafficking of polyadenylated RNA. Candidate disease gene, NXF5, encodes an mRNA export factor. Genetic deletion of fragile X mental retardation protein (FMRP) prevents its interaction with export factor, NXF2. Disease-causing mutations in the gene encoding mRNA export protein, Gle1. Nuclear retention of CUG repeat expanded DMPK mRNA and CCUG repeat expanded ZNF9 mRNA in DM1 and DM2, respectively.

Reference(s) Kawahara et al. (2004)

Vollmar et al. (2004) Vissel et al. (2001) Gurevich et al. (2002) Monani (2005)

(Neumann et al., 2006; Sreedharan et al., 2008; Vance et al., 2009) Xiao et al. (2008) Lin et al. (1998) Gasparini et al. (2007)

Ranum and Cooper (2006)

Young et al. (2005) Manabe et al. (2003) Myers et al. (2007)

Gnjec et al. (2008)

Nousiainen et al. (2008)

(Jin et al., 2007; Sofola et al., 2007) Buckanovich et al. (1993) Nakata et al. (2009) (Licatalosi and Darnell, 2006; Ranum and Cooper, 2006) Lim et al. (2008) Abu-Baker and Rouleau (2007) Abu-Baker and Rouleau (2007) Jun et al. (2001) Zhang et al. (2007)

Nousiainen et al. (2008) Ranum and Cooper (2006)

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Table 1 (continued) Neurological disorder AD mRNA FXS stabilisation and localisation SMA ALS

DM

Comments

Reference(s)

Protective polymorphism in the UAP56 promoter and upregulated mRNA levels. Trinucleotide repeat expansion in the 5'UTR of the FMR1 gene prevents normal FMRP expression.

Gnjec et al. (2008)

Affected localisation of β-actin mRNA to the growth cones of motor neurones. TDP-43 stabilises human low molecular weight neurofilament (hNFL) mRNA by directly binding to the 3'UTR. Sequestration of MBNL2 may interfere with the localisation of integrin α3 mRNA to adhesion complexes.

Rossoll et al. (2003)

neurones to AMPA receptor-mediated excitotoxicity (Van Den Bosch et al., 2006). The calcium permeability of the GluR2 receptor subunit is regulated by a virtually 100% efficient Ato-I RNA editing event which converts a glutamine to an arginine (the Q/R site) in a transmembrane domain of the protein (Sommer et al., 1991). Defective RNA editing at the Q/R site results in neuronal death which can be rescued by the restoration of RNA-editing, thus efficient RNA editing of the GluR2 receptor subunit is important for neuronal survival (Higuchi et al., 2000). In ALS, the editing efficiency of the GluR2 receptor subunit Q/R site is specifically reduced to between 0% and 100% in spinal motor neurones compared with controls (Kawahara et al., 2004). However, in this study RNA editing of the GluR2 Q/R site was not affected in upper motor neurones which are also affected in ALS suggesting that there could be another underlying cause such as oxidative stress (Rothstein, 2009). Evidence that an increased calcium permeability of the GluR2 receptor subunit is a plausible cause for sporadic ALS comes from transgenic mice expressing a GluR2 subunit with an N residue at the Q/R position; these mice suffer a late-onset ALS-like phenotype (Kuner et al., 2005). Small disturbances in the RNA editing efficiency of the Q/R GluR2 site have also been observed in the prefrontal cortex of Alzheimer's disease (AD) and schizophrenic patients as well as in the striatum of patients with Huntington's disease (HD) (Akbarian et al., 1995). A-to-I RNA editing at another GluR2 site converting an arginine to glycine (R/G) alters the desensitisation kinetics of receptors containing edited subunits (Lomeli et al., 1994). An increase in GluR2 editing at this R/G site in the hippocampus of patients with epilepsy has been observed (Vollmar et al., 2004). In fact, altered RNA editing has been well documented in epilepsy. Besides GluR2, the GluR6 subunit of kainate (KA) receptors also undergoes Q/R site editing that reduces the calcium permeability of the receptor; editing at this site is thought to modulate synaptic plasticity and seizure vulnerability (Morabito and Emeson, 2009; Vissel et al., 2001). In addition to glutamate receptors, altered RNA editing of the serotonin 5-HT2C receptor has been reported in depressed suicide victims as well as in response to antidepressant medication (Gurevich et al., 2002; Niswender et al., 2001). Thus in addition to neurodegenerative conditions such as ALS, aberrant RNA editing also plays a role in epilepsy and affective disorders such as depression and schizophrenia.

3.2.

(Oberle et al., 1991; Verkerk et al., 1991)

Strong et al. (2007)

Adereth et al. (2005)

Alternative splicing

As a result of the diversity generated by alternative splicing and its use to fulfil the physiological requirements of the cell, the disruption of its control can lead to cellular dysfunction and disease. In fact it is estimated that up to 50% of diseasecausing mutations affect pre-mRNA splicing in humans (Cartegni et al., 2002), these mutations can either hinder the splicing of a single gene through the disruption of regulatory cis-elements or have a trans-acting effect on the splicing of multiple genes by disrupting components of the spliceosome. Many excellent reviews have documented aberrant alternative splicing in neurodegeneration (Gallo et al., 2005; Licatalosi and Darnell, 2006), these are summarised in Table 1 with a couple of examples detailed below. Spinal muscular atrophy (SMA) is a childhood motor neurone disease characterised by the deletion of the survival for motor neurone gene 1 (SMN1) (Monani, 2005). This loss cannot be compensated for by its paralog SMN2, which differs by a single nucleotide in exon 7 thought to either create an exonic splicing silencer element or disrupt an existing exonic splicing enhancer element (Cartegni and Krainer, 2002; Kashima and Manley, 2003; Monani, 2005). Either way, the skipping of exon 7 is enhanced resulting in the production of a truncated, unstable form of the SMN protein. SMN is a ubiquitously expressed splicing factor required for the assembly of snRNPs (forming the so called SMN complex) but SMN deficiency selectively affects motor neurones. This deficiency results in cell-type- and snRNA-specific effects giving rise to a different set of snRNPs in SMN deficient cells (Monani, 2005; Zhang et al., 2008). This causes numerous widespread cell-typespecific splicing defects that alter the mRNAs of many functionally diverse genes (Zhang et al., 2008). Therefore, SMA can be considered a general splicing disease twice inflicted by defects in RNA processing, namely the skipping of SMN exon 7 and the loss of SMNs function as a splicing regulator. The motor neurone-specific effect may be a result of one or more aberrantly spliced transcripts or the combined effect of a number of splicing defects (Zhang et al., 2008). Mutations in splicing factor sequence can also lead to disease. Two such cases have revolutionised the ALS field firmly establishing ALS as a disease of RNA metabolism. Disease

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causing mutations in the genes encoding the RNA-binding proteins, TDP-43 and FUS, have been identified in familial ALS (Kwiatkowski et al., 2009; Lagier-Tourenne and Cleveland, 2009; Sreedharan et al., 2008; Vance et al., 2009). TDP-43 is additionally a major component of the ubiquitinated inclusions characteristic of sporadic ALS (Neumann et al., 2006) and furthermore, inclusions positive for either TDP-43 or FUS are present in a growing number of neurodegenerative disorders (Doi et al., 2010; Kwong et al., 2008; Woulfe et al., 2010). Both TDP-43 and FUS are splicing regulators (Lagier-Tourenne and Cleveland, 2009) although precise neuronal functions have not been identified; TDP-43 has been shown to promote the inclusion of SMN exon 7 and stabilise human low molecular weight neurofilament (hNFL) mRNA (Bose et al., 2008; Strong et al., 2007). With the exception of a mutation located in an RNA recognition motif, all TDP-43 mutations identified to date are located in the C-terminal domain of the protein which is important for interactions with members of the hnRNP family of splicing regulators (Buratti et al., 2005; D'Ambrogio et al., 2009; Kabashi et al., 2008; Lagier-Tourenne and Cleveland, 2009). It remains an important goal to determine whether TDP-43 or FUS mutations or indeed their sequestration to inclusions disrupts RNA metabolism.

3.3.

Polyadenylation

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is a progressive adult onset disorder characterised by dysphagia, ptosis and proximal limb weakness (Davies et al., 2006). Whilst primarily a myopathic disorder, most OPMD homozygote patients suffer cognitive decline, depression and psychosis (Abu-Baker and Rouleau, 2007; Blumen et al., 2009). OPMD is caused by a GCG repeat expansion in the coding region of polyadenylation-binding protein nuclear 1 (PABPN1, also known as PABP2). This expansion is translated into an Nterminal polyalanine (polyA) tract usually 12–17 amino acids long in patients; the mutant protein aggregates into filamentous intranuclear inclusions in skeletal muscle fibers, the pathological hallmark of OPMD (Davies et al., 2006). These inclusions have also been found in neuronal cells of transgenic OPMD mice and in post mortem brain sections of an OPMD patient (Abu-Baker and Rouleau, 2007). PABPN1 is a predominantly nuclear protein mostly concentrated in speckles, dynamic subnuclear structures enriched in RNA processing proteins. PABPN1 binds to poly(A) tails stimulating progressive polyadenylation forming part of the complex that tethers poly (A) polymerase to the 3′ end of mRNA (Abu-Baker and Rouleau, 2007; Wahle, 1991). As with many RNA processing proteins, PABPN1 is multifunctional with a secondary role in nuclear mRNA export (Apponi et al., 2010). Although most evidence points to a toxic gain-of-function mechanism (Abu-Baker and Rouleau, 2007; Davies et al., 2006), the role of intranuclear inclusions in OPMD pathogenesis is debated and a loss of function mechanism cannot be excluded. Surprisingly, the poly(A) tail length of mRNAs appears unaffected in OPMD myoblasts (Calado et al., 2000). However, high concentrations of poly(A) RNA are sequestered to intranuclear inclusions in OPMD (Calado et al., 2000). As a result, the trafficking of poly(A) RNA may be disrupted which could ultimately lead to lowered protein expression. Further evidence for an

impairment of RNA processing in OPMD comes from the sequestration of PABPN1-binding proteins, hnRNPA1 and A/B, to intranuclear inclusions identified in both cell models and OPMD patient tissue (Fan et al., 2003). The importance of PABPN1 in RNA processing and myogenesis was recently demonstrated using siRNAs in primary mouse myoblasts. Depletion of PABPN1 results in a shortening of poly(A) tails, nuclear accumulation of poly (A) mRNAs and impaired myoblast differentiation and proliferation strengthening the loss-of-function hypothesis (Apponi et al., 2010). It is curious therefore that a shortening of poly(A) tails was not observed in the Calado et al. study; they analysed poly(A) tails of up to 180 nt in length whilst in PABPN1 depleted primary myoblasts, only the shortening of poly(A) tails 200–300 nt long was observed, thus further study is required to ascertain whether polyadenylation is disrupted in OPMD muscle tissue (Abu-Baker and Rouleau, 2007). Whilst it may remain uncertain whether polyadenylation itself is affected, the aggregation of PABPN1 likely results in a knock-on effect disrupting several RNA processing events once again highlighting their exquisite coupling to one another.

3.4.

Nuclear mRNA export

X-linked mental retardation (XLMR) encompasses hundreds of disorders characterised by an intellectual disability and limitations in adaptive behavior, these include fragile X syndrome (FXS), the most common form of inherited mental impairment (Chiurazzi et al., 2008). The nuclear mRNA export factor (NXF5) gene has been identified as a candidate disease gene for XLMR (Jun et al., 2001). In addition, FXS is caused by a loss of the fragile X mental retardation (FMRP) protein which has been indirectly implicated in mRNA export through its interaction with NXF2 (Zhang et al., 2007). FMRP is an RNA-binding protein with unclear functions, it is implicated in the regulation of translation and its loss alters synaptic plasticity (Jin and Warren, 2003). The foetal motor neurone disease, lethal congenital contracture syndrome type 1 (LCCS1) characterised by foetal immobility and a loss of anterior horn neurones, is an autosomal recessive disease caused by mutations in GLE1 which encodes a required mediator of nuclear mRNA export, Gle1 (Nousiainen et al., 2008). Mutations in GLE1 also cause lethal arthrogryposis with anterior horn cell disease (LAAHD), another rare fatal foetal anterior horn disorder (Nousiainen et al., 2008). On the cytoplasmic face of the NPC, Gle1 binds to and activates the DEAD box RNA helicase Dbp5 increasing its catalytic efficiency. Dbp5 releases bound export proteins from the newly emerged transcript for recycling back to the nucleus (Hurt and Silver, 2008). Coincidently, the activity of Gle1 is positively regulated by soluble inositol hexakisphosphate (InsP6) and mutations in HER3 and PIP5K1C, genes involved in the synthesis of InsP6, cause LCCS types 2 and 3 respectively (Hurt and Silver, 2008; Narkis et al., 2007a,b). As well as mutations in mRNA export factors, the nuclear retention of mRNA as a result of sequestration to nuclear inclusions also occurs. For example, the characteristic inclusions of OPMD contain trapped mRNA as well as factors involved in nuclear export such as polyA expanded PABPN1 (Abu-Baker and Rouleau, 2007). In fact, protein aggregation as a result of trinucleotide repeat expansion is a well documented phenomenon

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and a classical example of nuclear RNA retention comes from the polyglutamine diseases. Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the 3'UTR of the DMPK gene; patients suffer an ultimately lethal muscle degeneration and myotonia. As with OPMD, DM1 also involves secondary CNS pathology (Ranum and Cooper, 2006). In DM1, CUG expanded RNA is retained in the nucleus in punctate foci where it disrupts both nuclear and cytoplasmic RNA processing events. Similarly, DM2 is caused by CCUG repeats in intron 1 of the zinc finger protein-9 (ZNF9) gene resulting in RNA foci and nuclear retention of expanded RNA (Ranum and Cooper, 2006). DM is thus caused by a trans-dominant pathogenic role of RNA, the splicing factor muscleblind like 1 (MBNL1) is sequestered to the double stranded hairpin structure formed by CUG repeats affecting several muscle-related splicing events which have been correlated with disease symptoms (Ranum and Cooper, 2006). In addition, the steady-state level of CUG-binding protein 1 (CUGBP1 – a member of the CELF family of splicing factors) is increased in muscle tissue from DM patients (Kuyumcu-Martinez et al., 2007). Besides disruptions in splicing, the inappropriate binding of proteins to CUG repeat expanded mRNA and its retention in the nucleus points to aberrations in the export of expanded mRNAs. Indeed DMPK protein levels are decreased in DM1 myoblasts (Furling et al., 2001) and defects in the export protein Aly aggravate the disease phenotype in a DM1 fly model (GarciaLopez et al., 2008). In AD, a protective role for the nuclear mRNA export and splicing factor, UAP56, has been suggested. UAP56 is an ATPdependant RNA helicase of the DEAD box family involved in spliceosome assembly and mRNA export where it recruits Aly to mRNA through a direct interaction. The importance of UAP56 is demonstrated by the removal of its ATP-binding activity which strongly inhibits mRNA export (Kota et al., 2008; Luo et al., 2001). UAP56 negatively regulates the production of pro-inflammatory cytokines associated with AD (IL-1, IL-6 and TNFα) and UAP56 mRNA levels are elevated in AD brains (Allcock et al., 2001; Wong et al., 2003). Furthermore, a polymorphism in the promoter region has been associated with a reduced risk of AD (Gnjec et al., 2008). Whether these phenomena are related to the role of UAP56 as an export or splicing factor has not been investigated.

3.5.

mRNA localisation and stabilisation

The intricately coupled processes of mRNA stabilisation, localisation and translation can be misregulated through the aberrant binding of proteins to the 5’ and 3'UTRs. For example, a CGG triplet repeat expansion of >200 nt in the 5'UTR of the FMR1 gene causes FXS where the expansion results in a loss of function due to the prevention of normal expression of FMRP (Oberle et al., 1991; Verkerk et al., 1991). Some carriers of FMR1 premutation alleles with only 55–200 repeats develop a distinct syndrome termed fragile X-associated tremor/ataxia syndrome (FXTAS) characterised by tremor, ataxia, dementia, parkinsonism and autonomic dysfunction (Hagerman and Hagerman, 2004). In FXTAS, the overexpression of the premutant mRNA results in the sequestration of RNA-binding proteins including Pur α and CUG-BP (Jin et al., 2007; Sofola et al., 2007). The SMN protein complexes with hnRNP R to regulate the localisation of β-actin mRNA to the growth cones of motor neurones (Rossoll et al., 2003). In a mouse model of SMA,

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motor neurones have a reduced axonal growth which is correlated with reduced β-actin protein and mRNA levels in the distal region of axons and growth cones thus implicating aberrant mRNA localisation in SMA (Rossoll et al., 2003). In addition to the role of MBNL proteins in alternative splicing, MBNL proteins also function in the regulation of mRNA localisation. MBNL2 co-localises with integrin α3 mRNA in adhesion complexes of the cytoplasm of epithelial cells. It is also physically associated with the 3'UTR of integrin α3 mRNA; the knockdown of MBNL2 abolishes the localisation of integrin α3 to adhesion complexes demonstrating the involvement of MBNL2 in the localisation of integrin mRNA (Adereth et al., 2005). MBNL2 is also sequestrated to RNA foci in DM (Miller et al., 2000) and may therefore interfere with its role in mRNA localisation and well as splicing. In ALS, hNFL mRNA levels are decreased in degenerating spinal motor neurones and TDP-43 has been identified as an hNFL mRNA stabilising protein through its direct interaction with hNFL 3'UTR (Strong et al., 2007). In fact TDP-43 may also play a role in mRNA localisation due to its presence in RNA granules from the developing brain (Elvira et al., 2006). The stabilisation and localisation of mRNA may therefore be misregulated in ALS as a consequence of the mislocalisation and aggregation of TDP-43. TDP-43 also acts as a translational repressor in vitro (Wang et al., 2008) and is reportedly involved in microRNA biogenesis via interactions with the Drosha complex (Buratti and Baralle, 2008; Gregory et al., 2004). An interesting link between motor neurone pathology and mRNA stabilisation, localisation and translation comes from the multimeric protein, eukaryotic elongation factor 1 (eEF1). Whilst primarily involved in protein synthesis, it has long been suggested that the eEF1A subunit has functions in the cell beyond translation such as in cytoskeletal dynamics; this could account for the unusually high abundance of eEF1A compared to other elongation factors (Condeelis, 1995; Liu et al., 1996). The eEF1A subunit is a known 3'UTR-binding protein involved in the localisation of a number of transcripts (Liu et al., 2002; Mickleburgh et al., 2006). eEF1A is found in a complex with the transcription factor ZPR1, the interaction of ZPR1 with SMN is vital for the correct localisation of SMN and evidence suggests that eEF1A is present in such an ZPR1/SMN complex (Abbott et al., 2009). Two isoforms of eEF1A, eEF1A1 and eEF1A2, share a sequence identity of >90% but have different expression patterns with the latter being detected only in the heart, brain and muscle (Knudsen et al., 1993). Interestingly, eEF1A2 is implicated in motor neurone pathology. In the wasted mouse, the gene encoding eEF1A2 is deleted by a spontaneous autosomal recessive mutation, wasted (wst) (Newbery et al., 2005; Shultz et al., 1982). Mice homozygous for this mutation grow normally until post natal day 21 when they develop tumours, abnormal gait and weight loss due to a loss of muscle bulk leading to death by about 28 days (Shultz et al., 1982). The lack of eEF1A2 in these mice results in a progressive loss of motor neurone function due to a retraction of motor nerve terminals in muscle demonstrating that eEF1A2 expression is required for the maintenance of neuromuscular junctions (Newbery et al., 2005). During postnatal development, eEF1A1 expression declines until it is undetectable by 21 days in the brain, heart and skeletal muscle; thus eEF1A2 becomes the major form in maturity (Pan et al., 2004).

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As expected, neither isoform is present in wasted mice after the developmental switch suggesting that the silencing of eEF1A1 remains functional in these mice. This developmental switch therefore coincides with the onset of muscle wasting and neurological impairment in wasted mice (Pan et al., 2004).

4.

Concluding remarks

It has become increasingly apparent that aberrant RNA processing plays a role in a wide spectrum of neurological diseases. Whilst disorders such as SMA and DM have long been established as diseases of RNA metabolism, a definite role for aberrant RNA processing in the pathogenesis of other diseases is emerging. In the case of ALS, aberrations in several RNA processing events could account for both familial and sporadic forms of the disease firmly establishing ALS as a disease of RNA processing. Indeed, the simultaneous disruption of a number of RNA processing events could contribute to the pathogenesis of several disorders due to their extensive coupling to one another. RNA processing should therefore be considered a prime target for therapeutic intervention.

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