The RNA-binding protein HuD:a regulator of neuronal differentiation,maintenance and plasticityJulie Deschenes-Furry,1 Nora Perrone-Bizzozero,2 and Bernard J. Jasmin1,3*

SummarymRNA stability is increasingly recognized as beingessential for controlling the expression of a wide varietyof transcripts during neuronal development and synapticplasticity. In this context, the role of AU-rich elements(ARE)containedwithin the30 untranslated region (UTR)oftranscripts has nowemerged as key because of their highincidence in a large number of cellular mRNAs. Thisimportant regulatory element is known to significantlymodulate the longevity of mRNAs by interacting withavailable stabilizing or destabilizing RNA-binding pro-teins (RBP). Thus, in parallel with the emergence of ARE,RBP are also gaining recognition for their pivotal role in

regulating expression of a variety of mRNAs. In thenervous system, the member of the Hu family of ARE-binding proteins known as HuD, has recently beenimplicated in multiple aspects of neuronal functionincluding the commitment anddifferentiation of neuronalprecursors as well as synaptic remodeling in matureneurons. Through its ability to interact with ARE andstabilize multiple transcripts, HuD has now emerged asan important regulator of mRNA expression in neurons.The present review is designed to provide a comprehen-sive and updated view of HuD as an RBP in the nervoussystem. Additionally, we highlight the role of HuD inmultiple aspects of a neuron’s life from early differentia-tion to changes in mature neurons during learningparadigms and in response to injury and regeneration.Finally, we describe the current state of knowledgeconcerning the molecular and cellular events regulatingthe expression and activity of HuD in neurons.BioEssays28:822–833, 2006. � 2006 Wiley Periodicals, Inc.

Introduction

In recent years, the role of the 30untranslated region (30UTR)

has become increasingly recognized in modulating mRNA

stability, localization and translation in several cell biological

aspects. More specifically, the 30UTR can control mRNA

expression at the earliest stages of oogenesis, embryogen-

esis and tissue development (see for review Refs 1,2). Post-

transcriptional events have been implicated in the regulation of

short-lived mRNAs encoding specific cytokines, growth-

factors and immediate-early response genes (see for review

Ref. 3). Importantly, post-transcriptional mechanisms are not

limited to short-lived transcripts since they are also important

for the control of variousmRNAs encoding proteins involved in

metabolic activities including for example, the regulation of

parathyroid hormone mRNA stability by calcium and phos-

phate.(4) In parallel to the discovery of additionalmRNAs being

regulated via post-transcriptional mechanisms, there is also

an increase in the number of diseases of the cardiovascular,

immune and nervous systems that are associated with

mutations in the 30UTR of specific mRNAs or with misregula-

tion of RNA-binding proteins (RBP) that interact with 30UTRs

(see for review Refs 3,5–8).

Within the nervous system, the role of RBP in all aspects of

neuronal function is not only becoming evident but is also

1Department of Cellular and Molecular Medicine and Centre for

Neuromuscular Disease, Faculty of Medicine, University of Ottawa,

Ottawa, Ontario, Canada.2Department of Neurosciences, University of New Mexico, School of

Medicine, Albuquerque, New Mexico.3Molecular Medicine Program, Ottawa Health Research Institute,

Ottawa, Ontario, Canada.

Funding agencies. Work in the Jasmin laboratory is supported by the

Canadian Institutes of Health Research, the Muscular Dystrophy

Association of America and the Association Francaise contre les

Myopathies. Work in Perrone-Bizzozero’s laboratory is supported by

the National Institutes of Health; Grant number: NS30255.

*Correspondence to: Bernard J. Jasmin, Department of Cellular and

Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa,

ON, K1H 8M5. E-mail: jasmin@uottawa.ca

DOI 10.1002/bies.20449

Published online in Wiley InterScience (www.interscience.wiley.com).

822 BioEssays 28.8 BioEssays 28:822–833, � 2006 Wiley Periodicals, Inc.

Abbreviations: 30UTR, 30 untranslated region; AChE, acetylcholines-

terase; ARE, AU-rich element; CARM1, co-activator associated

methyltransferase 1; CPEB, cytoplasmic polyadenylation element

binding; DRG, dorsal root ganglion; ELAV, embryonic lethal abnor-

mal-vision; FMRP, Fragile-X mental retardation protein; hnRNP A1 or

K, heteronuclear ribonucleoprotein A1 or K; IMP1, insulin-like growth

factor mRNA binding protein 1; KSRP, K homology-type splicing

regulatory protein; MARCKS, myristoylated alanine-rich C kinase

substrate; PABP, poly(A)-binding protein; RBP, RNA-binding protein;

RNP, ribonucleoprotein; RRM, RNA-recognition motif; SCG, superior

cervical ganglion; SNP, single nucleotide polymorphism; ZBP, zipcode-

binding protein.

Review articles

gaining importance. This is likely related to the fact that

amongst cell types, neurons present an architectural chal-

lenge because of their relatively small cell body size and

extensive network of projections and connections. Aswe learn

more about the basic mechanisms underlying neurogenesis,

neurite elongation, synapse formation and plasticity, we find

that RNA-binding proteins are not only involved in all these

events but that they are in fact essential and, hence, act as

key components, to ensure their appropriate unfolding and

completion. Consequently, it is not surprising to find direct

associations between neurodegenerative diseases and con-

ditions such as spinal muscular atrophy, amyotrophic lateral

sclerosis and Fragile X syndrome, and perturbation in RNA

regulation and/or RBP activity (see for review Refs 3,5,6,8,9).

There are a number of well-characterized RBP that have

been shown to assume specific roles in normal neuronal

development and function (see Table 1).(10–13) Among

these RBP, the cytoplasmic polyadenylation element binding

(CPEB) protein, zipcode-binding protein (ZBP), Fragile-Xmental

retardation protein (FMRP) and Staufen are important for

mRNA transport along dendrites or axons.(14–19) In addition to

transporting specific target transcripts, CPEB, ZBPandFMRP

all have translational regulatory roles such that translation is

inhibited during transport and activated at the final destination

by specific signals.(20–24) Besides regulating transport and

translation, RBP are known to control mRNA degradation.

To date, the majority of these, including specifically AUF1

(seeTable 1), havebeen shown to stimulate or facilitatemRNA

decay.(25,26) In comparison, theHu family of RBP, ofwhichHuD

is the focus of this review, are among the few known proteins

that stabilize transcripts in the cytoplasm.

Over the last 15 years, there has therefore been grow-

ing interest in the function and regulation of the Hu family of

RBP, which is directly involved in development of paraneo-

plastic encephalomyelitis and sensory neuronopathy syn-

dromes.(31,33,36) These syndromes result from the expression

of neuronal proteins by tumor cells in the body, typically small-

cell lung cancer tumors. Ectopic expression of these ordinarily

immune-privileged proteins results in production of onconeur-

al antibodies that attack the nervous system and lead to the

development of autoimmune neurologic diseases. The anti-

body produced in the above disorder was termed anti-Hu

based on the name of the patient in which the antibody was

discovered.(31,37) The Hu antibody was used to identify HuD

and other Hu proteins (see further).(33)

Amongst the different family members, HuD is recognized

as oneof the earliestmarkers of neuronal cells aswell as being

an essential regulator of neuronal differentiation and survi-

val.(8,27,38–43) Of particular interest and of potential clinical

relevance is the recent association between polymorphisms

in the HuD gene and age-at-onset of Parkinson’s disease.(44)

A total of nine single nucleotide polymorphisms (SNP) within

the human HuD locus region were genotyped in this study,

of which two, one in intron 2 and one in exon 8, showed

Table 1. Neuronal RNA-binding proteins

RBP Domains Function and recognized element

Hu (HuB, HuC and HuD) . 3 RRM . mRNA stabilization

. Cell-cycle regulation and neurite extension

. ARE (AUUUA, see text)(8,27–31)

AUF1 (p37, p40, p42, p45) . 2 RRM . mRNA destabilization

. ARE(25,26,32)

Cytoplasmic polyadenylation element binding (CPEB) protein . 2 RRM . Polyadenylation and translation activation (derepression)

. mRNA dendritic transport

. Synaptic plasticity

. CPE (UUUUUAU)(18,20,23)

Musashi (Msi1 and 2) . 2 RRM . Translation repression

. Neuronal development(10,12)

Zipcode-binding protein (ZBP1 and 2) . 1 RRM . mRNA transport

. 4 KH . Translation repression

. Growth-cone dynamics

. Zipcode(14,17,19,21,24)

Human K homology-type splicing regulatory protein (KSRP) or rat . 4 KH . mRNA dendritic transport

MARTA-1 . mRNA degradation(15,16,33)

NOVA-1 and NOVA-2 . 3 KH . Splicing regulation

. Neuronal survival

. UCA(U/C) stem-loop repeats(8,11,31)

Fragile X mental retardation protein (FMRP) . 2 KH . Translation repression and mRNA transport

. 1 RGG . Synaptic structure and plasticity

. G quartet structure(8,34,35)

Staufen (Stau1 and 2) . 5 dsRBD . Dendritic mRNA targeting

. Double-stranded RNA(13)

Review articles

BioEssays 28.8 823

moderately to strongly significant effects on age-at-onset of

Parkinson’s disease. Here, we therefore present a compre-

hensive and updated view of HuD and its role in multiple

aspects of a neuron’s life including the initial phenotypic

commitment and differentiation, as well as synaptic remodel-

ing during learning paradigms and in response to injury and

regeneration. Additionally, we describe the current state of

knowledge concerning the molecular and cellular events

regulating the expression, activity and function of HuD in

neurons.

The HuD Gene, its transcripts and

encoded proteins

As mentioned, HuD was the first of the Hu proteins to be

identified as theanti-Huantigen in patientswith paraneoplastic

encephalomyelitis and sensory neuronopathy.(31,33,36) The Hu

protein family also includes HuB (Hel-N1), HuC (Hel-N3) and

HuR (HuA).(33) Whereas HuR is ubiquitously expressed

and HuB is found in neurons and gonads, expression of HuC

and HuD is restricted to neurons. Due to the presence of three

highly conserved RNA-recognition motifs (RRM) (see below

and for review 45,46), HuD shares a high degree of homology

with the Drosophila proteins ELAV (responsible for the

Embryonic Lethal Abnormal Vision phenotype; see Fig. 1)

that is required for the normal development and maintenance

of the nervous system.(33,47)

HuD is encoded by a relatively large gene (�146 Kb)

located on chromosome1 in humans (1p34) and chromosome

4 in mice (49.5 cM). As shown in Fig. 2, some of the exons are

separatedby large intronic regions. The localizationof theHuD

gene in the mouse genome was based on syntenic regions

between human and mouse, suggesting that the flanking

genes are conserved between species.(48) The gene consists

of three putative non-coding exon 1 variants, termed 1a, 1b

and1c, andseveral commoncodingexons (2, 3, 4, 5and8; see

Fig. 2).(30,48) The first and second RRM are encoded by exons

2 and 3, and exons 4 and 5, respectively, while the third RRM is

encodedbyexon 8. TheHuDgene is also subject to alternative

splicing of exons 6 and 7 which affects the length of the

hinge region linking the second and third RRM. This results in

the three different transcripts and molecular forms, termed

HuDpro, HuD and HuDmex(30) of which HuDpro and HuD are the

major variants.(47–50) The resulting proteins have molecular

masses ranging between37and43 kDaandare characterized

by the three RRMs and unique nuclear export signals located

within the hinge region.(40)With theexceptionof theN-terminal

domain and the hinge region, the protein sequences between

the different Hu proteins are very similar (70 to 85% identity;

see Fig. 1).(47)

HuD’s RNA-recognition motifs (RRM)

and target transcripts

As shown in Figs 1 and 2, all Hu proteins contain three RRM.

RRMs are found in various proteins involved in RNA proces-

sing and turnover, including hnRNPA1 involved in splicing and

transport of RNA, poly(A)-binding protein (PABP) involved in

transcript stability and translation, and the Drosophila ELAV

protein involved in RNA splicing (see for review 45,46). The

Figure 1. The murine Hu family of ARE-

binding proteins shares a high degree of

homology with the Drosophila protein ELAV.

HuD, HuB, HuC, HuR and ELAV protein

sequences are aligned demonstrating the high

degree of homology especially within the RRM

(highlighted in yellow). RNPmotifs that bind the

RNA are indicated by underlined blue letters.

The exons encoding each protein region are

indicated above the sequence alignment and

the splicing between the exons is indicated by

the slanted bar. The underlined and red residue

(R236) corresponds to the methylated arginine

in HuD and HuR.

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824 BioEssays 28.8

RRM is a conserved structure of�80–90 residues containing

two consensus ribonucleoprotein (RNP) motifs separated by

25–35 amino acids that interact directly with the RNA. The

RNP motifs, octameric RNP-1 and hexameric RNP-2, each

contain three conserved aromatic residues that are implicated

in RNA interactions in a number of different RBP including

proteins that bind pre-mRNA, mRNA, pre-rRNA as well as

small nuclear and heteronuclear RNAs. The consensus

structure of RRM consists of b1-a1-b2-b3-a2-b4 and the

location of the RNPmotifs in the first and third b-strands of theRRM is highly conserved.(45,51) Although the structure of RRM

iswell preserved, only a few residues foundmostly in the RNP

motifs are highly conserved. Consequently, it is the variable

regions between the RNP motifs and RRM that tend to lend

sequence specificity to the RBP. Similarly to other RRM-

containing RBP, the first and second HuD RRM consist of the

consensus RRM structure and form a cleft between them

where the RNA is bound specifically between the b-sheets.(52)

Crystal structureexperiments revealed that the residueswithin

the first and second HuD RRM that interact with the RNA, are

conserved between HuD and ELAV proteins.(52)

Hu proteins recognize and bind specifically to well-

described AU-rich elements (ARE) found within the 30UTR of

approximately 1 in 20 human genes(53,54) and directly

implicated in RNA turnover.(30,53,55,56) The AREs are sepa-

rated into three classes based on their sequence and

structure. Class I AREs consist of one to three dispersed

AUUUAmotifs separated byU-rich regionswhile class II AREs

consist of multiple clusters of AUUUA motifs. Class III AREs,

although less-well defined, consist mainly of U-rich sequences

and do not contain the AUUUA pentamer.(57) The different

classes of AREs appear to instruct distinct modes of mRNA

decay. Class I and III ARE-directed mRNA degradation

involves simultaneous deadenylation of mRNAs resulting in

a pool of transcripts of homogeneous lengths, termed

distributive deadenylation, which is followed by degradation

of the mRNA body. In contrast, Class II ARE-directed mRNA

degradation consists of complete deadenylation of one

transcript resulting in a pool of mRNAs of heterogeneous

lengths, termed processive deadenylation.(58) Recently, HuD-

RNA crystal structure and in vitro binding assays have

determined that the consensus sequence recognized by

RRM1 and RRM2 is X-U/C-U-X-X-U/C?-U-U/C, where the

‘‘?’’ denotes questionable binding of theC in this location.(52,59)

Consequently, HuD has been demonstrated to bind and

stabilize transcripts with CU- and U-rich sequences as well

as class II and III AU-rich elements. The structure of the RRM

allows for the binding of any number of domains and RNA

conformations.(52) Similarly to HuR, HuD, while able to bind all

classes of ARE, retains some sequence specificity and

preference such that it will not bind a transcript based solely

on the presence of an ARE.(60)

Unlikemany other ARE-binding proteins that principally act

to destabilize transcripts, such as AUF1 family members

(Table 1), Hu proteins act to stabilize ARE-containing

transcripts thereby significantly prolonging their half-

life.(39,42,61,62) In vitro binding studies performed with HuD

have demonstrated that the RNA is bound primarily by the first

RRM while the second RRM, which also binds the RNA,

functions mostly to stabilize the RNA–protein complex.(52,63)

Figure 2. HuD gene, transcript and protein organization. A: HuD exon and intron organization is depicted. Green boxes correspond to

three putative alternative exon1 variants, blue boxes correspond to coding region exons and red boxes correspond to alternatively spliced

coding region exons. Note the large intronic regions separating exons 1 through 4. B: Alternative splicing of exons 6 and 7 results in the

production of three alternative transcripts (HuDpro, HuD and HuDmex) which have untranslated regions (UTR and exon 1 shown in green) of

varying and unknown lengths. The promoter region driving gene transcription and the predominant exon 1 variant alongwith the resulting 50

untranslated region, remain unknown.C:Protein organization of the differentHuD transcripts. Thevarying length of the hinge region linking

the second and third RRM results from alternative splicing of exons 6 and 7.

Review articles

BioEssays 28.8 825

The third RRM, in addition to participating in maintaining

RNA–protein complex stability, binds to poly(A) tails.(64–67) In

the latter case, the length of the poly(A) tail correlates with the

overall binding efficiency of Hu proteins.(64) In contrast, the

third RRM of mouse HuB and HuC could not bind to poly(A)

ribohomopolymers and generally showed very little RNA-

binding activity.(68) There are, however, some inconsistencies

between studies as another study performed with mouse HuC

demonstrated that the third RRM could specifically bind to

poly(A)-sepharose beads.(66) The different results obtained

with each Hu family member may simply reflect different

technical approaches. In that context, the studies performed

with HuD used complete in vitro transcribed GAP-43 tran-

scripts with differing lengths of poly(A) tails as opposed to

poly(A) nucleotide chains, and could consequently be con-

sidered a more accurate representation of the binding activity

of the third RRM.(64) Alternatively, it is possible that the third

RRM is also involved in protein–protein interactions rather

than direct RNA-binding, as suggested by Kasashima et al.

(2002), who showed that HuD, HuB and HuC could form

dimers via the third RRM.

As the variety of cis-acting elements recognized by the

RRM have increased, so have the number of transcripts that

interact with HuD. Accordingly, HuD has been shown to bind

and stabilize several developmentally regulated transcripts

including c-fos, c-myc, N-myc, p21waf1, neuroserpin and

MARCKS.(69–73) Interestingly, and in agreement with its role

in neuronal differentiation, many of the transcripts bound by

HuD play a key role in the formation of neuronal processes

such as GAP-43 and tau.(39,62,74) In this context, our studies

have recently shown that HuD also binds and stabilizes

acetylcholinesterase (AChE) transcripts during neuronal

differentiation.(42) Since in addition to its well-recognized

function in neurotransmission, AChE can also stimulate

neurite outgrowth (Refs 42,75,76 and references therein), it

appears therefore that HuD binds and regulates the stability of

a subset of transcripts with similar functional roles that is key

for neuronal development and plasticity.

Although the principal function of HuD is to stabilize its

target transcripts and increase their half-life, several studies

performed with HuD or other members of the Hu family

suggest that HuD is also involved in other aspects of mRNA

regulation (see Figure 3). The variable linker domain sepa-

rating the second and third RRMs has been suggested

to contain a novel nuclear export signal that allows HuD

to shuttle target transcripts out of the nucleus into the

cytoplasm.(29,40,77) In addition, the first and second RRM and

part of the linker domain have been shown to interact with the

primary mRNA export receptor TAP/NXF1 in cultured neuro-

nal cells.(78) In fact, previous studies have shown that neurite

elongation is severely impeded when shuttling of HuD is

blocked, suggesting that HuD plays an important role in

localizing its target transcripts to the cytoplasm.(40) Once in the

cytoplasm, HuD and its cargo are targeted to axons, in the

case of tau mRNA, and growth cones, in the case of GAP-43

mRNA.(79,80)

Specific subcellular localization of the HuD-mRNA gran-

ules appears to involve the KIF3A microtubule-associated

motor protein.(79) Along these lines, HuDwas shown to bind in

an RNA-dependent manner to insulin-like growth factor

mRNA-binding protein 1 (IMP1).(40,81) IMP1 is the mouse

ortholog of chicken ZBP1 which is known to have a role in

mRNA transport and translation regulation (see Table 1).(82)

Furthermore, HuD has also been shown to colocalize with

Figure 3. HuD function and regulation within

neurons. A: HuD gene transcription within

neurons may be controlled by neuron-specific

transcription factors. HuD transcript may also

be bound by itself. B: HuD binds its target

transcript within the cytoplasm and possibly

within the nucleus prior to nuclear export. Post-

translational modification of HuD, such as

methylation, inhibits HuD–RNA interactions.

C: HuD competes for target transcripts with

otherRBP that are predominantly destabilizing.

The abundance of the different RBP deter-

mines the eventual fate of transcripts. D: HuDstabilizes target transcripts and protects them

from decay. E: HuD binds to its target tran-

scripts in the axon hillock, along the axonand in

the growth cone. HuD transport may involve

molecular motors and interactions with other

RNA-transport proteins (see Table 1).

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826 BioEssays 28.8

ribosomes in dendrites of pyramidal neurons of the CA1 and

CA3 regions of the hippocampus.(83) Finally, HuD-bound

mRNAs are also targeted to ribosomes and polysomes,

similarly to HuB and neurofilament-M mRNA.(80,81,84) These

studies suggest therefore that HuD could also be involved,

either directly or indirectly through binding with other RBP

with translational functions, in stimulating translation of its

target transcripts. In this context, a recent study has shown

that HuB can interact with hnRNP K and both can bind to

p21 mRNA 30UTR.(85) This later work also demonstrated

that hnRNP K alone decreased the translation of this

transcript, whereas HuB could block this effect and indirectly

increase translation. Given the similarities in Hu protein

function and target transcripts, it is possible that HuD has

similar functions.

Functional role of HuD in neurons

The neuronal Hu proteins are known to be some of the earliest

markers of the neuronal phenotype since they appear to

suppress neuroblast proliferation and promote neuronal

differentiation.(47,68,86–88) In situ hybridization experiments

performed in developing and adult mouse and rat cerebral and

cerebellar cortex revealed a distinct expression pattern for

mRNAs encodingHuB, HuC andHuD.(47,89) During embryonic

development, HuD expression can be localized to cells exiting

the cell cycle in the ventricular zone, and to those migrating in

the intermediate zones and undergoing terminal differentia-

tion.(41,47,89) In the adult brain, HuD-positive neurons corre-

spond primarily to projection neurons found in the neocortex,

hippocampus, entorhinal cortex and cerebellum. Similarly,

HuD is very strongly expressed in the ventral motoneurons of

the spinal cord, the sensory neurons of the dorsal root ganglia

(DRG) and sympathetic neurons in their ganglia.(41,89) At the

subcellular level, HuD is detected in the cell body, axon and

growth cones.(47,79,80) Generally, HuB and HuD have a similar

pattern of expression which is somewhat different from HuC.

Although expression of HuB and HuD overlap, they appear to

have similar(68,85) and opposing(90,91) functions during neuro-

nal development depending on the context. For example, they

can both stimulate neuronal differentiation and neurite out-

growth but have opposing roles in progenitor cell self-renewal,

such that HuB positively and HuD negatively affects this

capacity.

Studies in which the expression level of HuD has been

manipulated byeither overexpression or downregulation, have

highlighted the significance of HuD in stabilizing various

mRNA in neurons. Specifically, HuD binds a subset of

transcripts that encode proteins involved in neuronal develop-

ment. Several recent studies have shown that overexpression

of HuD increases the rate and length of neurite outgrowth in

several different types of neuronal cells, including in both

primary cultures and neuronal cell lines. By contrast,

decreased HuD expression results in an inhibition of neurite

extension.(38,39,41,62) Together, these results indicate that HuD

is an important regulator of neurite elongation and morpholo-

gical differentiation.(8,92) Expression of HuD in non-neuronal

cells does not stimulate the development of processes,

suggesting that pluripotent cells must already be engaged

along a neuronal cell fate in order for HuD to be effective in

promoting morphological differentiation.(38,41) This effect is

specific to neuronal Hu proteins, as HuB and HuC can also

stimulatemorphological differentiation, while HuR is unable to

stimulate neurite extension in neural crest cells.(41,68,84)

Recently, HuD transgenic and HuD knockout mice have

beengeneratedand characterized.(91,93) HuD transgenicmice

specifically expressed higher total amounts of HuD in the

forebrain, particularly in all subregions of the hippocampus

including in dentate granule cells that do not normally express

HuD.(47,83,89) Interestingly, in both HuD-overexpressing and

-deficient mice, there are no apparent morphological or

structural defects in the adult brain. However, extension of

some cranial nerves is transiently impaired during embry-

ogenesis in HuD-deficient mice. In comparison, loss-of-

function alleles of Drosophila elav are embryonic-lethal and

hypomorphic mutations result in morphological and structural

defects of the eye.(94,95) Given that there are three neuronal Hu

proteins with similar functions and one ELAV, it is possible that

expression of the other members of the Hu family are

compensating for the loss of HuD in knockout animals.

Although there are no apparent morphological defects, HuD-

deficient mice display motor/sensory defects such as an

abnormal clasping reflex of the hindlimbs when suspended by

the tail.(91) The abnormal clasping reflex suggests a defect in

sensory and motor functions which is also confirmed by poor

performance in rotarod experiments.(91) Both of these mouse

models of aberrant HuD expression have initially been used to

confirm some of the in vitro and cell culture data and to further

elucidate the importance of HuD to neuronal development and

function.

During early developmental stages, HuD appears to be

involved at multiple levels of cell lineage commitment,

differentiation and survival. Studies using HuD knockout mice

showed that, during embryogenesis, the absence of HuD is

related to an increase in the progenitor cells’ ability to renew,

and to a decrease in their ability to exit the cell cycle and

morphologically differentiate into neurons.(91) Cells that are

not leaving the cell cycle are also more likely to undergo

apoptosis since levels of apoptosis are elevated in HuD-

deficient mice. In adult mice, the number of subventricular

zone progenitor cells is greater in the HuD knockout mice.

These studies suggest therefore, that HuD is important for

promoting the exit from the cell cycle, for negative regulation of

proliferation and stimulation of the differentiation process.(91)

In addition, an increased level of HuD in cultures of neural crest

cells precipitates neurotrophin dependence.(41,96) Similarly,

HuD overexpression in mouse embryonic stem cells can

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BioEssays 28.8 827

double the number of cells with long neurites and expressing

GAP-43 only when the cells are induced to differentiate with

retinoic acid.(38) The results obtained with HuD knockout mice

and from cultured neuronal cell lines, together with the

preferential expression of HuD in projection neurons, strongly

suggest that HuD through its stabilizing effect on several

transcripts, is essential for the proper development and

maintenance of neuronal extensions.

The significance of HuD in the regulated expression of

mRNAs and proteins involved in the growth and formation of

neuronal projections, suggests that it is also important to other

aspects of axonal and dendritic functions in the adult nervous

system.A fewdistinct andcomplementary studieshaveclearly

demonstrated that spatial learning tasks can enhance HuD

expression in hippocampal neurons resulting in a concomitant

increase in GAP-43 transcript levels.(97,98) Another experi-

mental paradigm, single trial contextual fear conditioning, also

demonstrated an association between HuD expression and

learning.(83) In the latter study, HuD expression was increased

in different regions of the hippocampus namely, in the hilar

region of the dentate gyrus, as well as in the CA3 and CA1

regions. Along similar lines, increased expression of HuD in

the hippocampus of transgenic mice overexpressing HuD,

stimulates GAP-43 mRNA expression and stabilization(93) as

well as AChE mRNA expression.(99) More specifically, over-

expression of HuD in dentate granule cells, where HuD is not

normally expressed, results in a marked increase in GAP-43

mRNA levels via post-transcriptional mRNA stabilization of

newly synthesized, but normally degraded, transcripts.(93)

Consequently, atypical expression of HuD in neurons may

have important regulatory effects on the expression of its

target transcripts and, thus, a significant downstreameffect on

neuronal function.

Given its significant role in morphological differentiation,

HuD has also been suggested to have a role in axonal

regeneration. Recent findings have shown that following nerve

crush of DRG sensory neurons and facial motoneurons, HuD

protein and transcript levels increasewithin 7 days of the injury

and remain elevated for up to 21 days.(100,101) This increase in

HuD expression is accompanied by a dramatic increase in the

mRNA levels of GAP-43, a known target of HuD. In

regenerating neurons of the DRG, HuD protein colocalized

with GAP-43 transcripts as well as with ribosomal RNA.(100) A

recent study has also demonstrated the co-localization of HuD

and GAP-43 mRNA in growth cones.(80) In this context, we

have found that exogenousexpressionof humanHuD in the rat

superior cervical ganglion (SCG) results in themaintenance of

AChE and GAP-43 mRNA levels following axotomy (unpub-

lished data). Together, these findings demonstrate that,

in addition to its role during neuronal development, HuD

expression and function correlate with plasticity of the nervous

system both in response to injury and during learning. Further

studies performed with the HuD-transgenic and -deficient

mice are necessary to strengthen these correlations into direct

or indirect involvement of HuD.

Molecular events regulating expression

of HuD and its function

Despite the well-established involvement of HuD in the control

of a subset of neuronal transcripts involved in neuronal

differentiation and plasticity, there appears to be a lack of

information concerning thenatureof themolecular andcellular

mechanisms that regulate expression of HuD in neurons. This

is important since a number of recent studies point to mRNA

stability as a consequence of functional antagonism between

stabilizing and destabilizing proteins (see for reviewRef. 102).

This is best exemplified by studies performed with the

ubiquitous Hu family member HuR, whose tissue expression

overlaps with AUF1, a family of four isoforms that are

predominantly destabilizing (Table 1).(32,103) In addition, many

transcripts that havebeen identifiedas targets forHuRarealso

AUF1 targets, such as p21, c-fos, TNF-alpha and cyclin

D1.(102,104) The antagonistic effects of these proteins have

been demonstrated using siRNA to individually decrease the

expression of HuR or AUF1, resulting in increased association

of the target transcript with the opposite protein and

subsequent decreased or increased stability, respectively.(104)

Using a similar approach, competition for transcript-binding

siteswas also demonstratedwithin the AUF1 family.(105) In this

context, it is important to note that AUF1 has also been

reported to have mRNA-stabilizing activity.(106,107) Thus,

it appears that competition for target transcripts and the

eventual outcome for their longevity are largely determined by

the relative abundance of the different RBP.(102,104) Accord-

ingly, it becomes important to gain a better understanding of

the events presiding over HuD expression in neurons.

Transcriptional and post-transcriptionalregulatory mechanisms.The HuD gene is divided into eight coding exons beginning

with exon 2 (see Figs 1 and 2).(48) A comparison of the

available cDNA sequences in GenBank (NM_010488,

BC052451 and BC052451) shows the presence of three

alternate non-coding exon1 splice variants termed exon1a, 1b

and 1c (see Fig. 2).(48) Presently, it is unclear whether a single

promoter is responsible for transcription of this large gene

with splicing of exon1 variants occurring subsequently or

whether there are multiple promoter choices driving specific

expression of exon 1 variants. Although the HuD 50 regulatory

region of the mammalian gene has yet to be isolated and

characterized, studies performed with the Xenopus laevis

HuD homologue, elrD, have shown that there are two possible

promoter regions upstream of exon 2.(108) The most 50

promoter region is upstream of a non-coding exon 1 and the

second promoter region is located within the first intron. Both

Review articles

828 BioEssays 28.8

promoter regions have been shown to drive transcription in a

neuronal-dependent fashion and to contain neuron-specific

transcription factor binding sites (E-box and neuron-restrictive

silencer factor site). Similarly, characterization of the human

HuB 50 regulatory region demonstrates the presence of cis-

acting elements that can direct tissue-specific expression in

neurons.(109) Analysis of the zebrafish HuC 50 regulatory

region resulted in the identification of multiple E-box se-

quences, a putative MyT1-binding site and two GC boxes that

could also drive neuron-specific expression of HuC.(110,111)

The Drosophila ELAV 50 flanking region including enhancer

domains within an intronic region, also confers neural-specific

expression patterns to this gene.(112) Accordingly, the specific

and early neuronal expression of HuD alongwith the presence

of alternate 50 regulatory regions strongly suggest that

expression of HuD is regulated by transcriptional mechanisms

involving selective cis-acting elements and trans-acting

factors that are specific to neurons (seeRef. 87). In agreement

with this view, a recent study demonstrated that the transcrip-

tional rate of HuD is significantly altered in neurons subjected

to changes in the levels of thyroid hormone.(113)

In addition to transcriptional regulatory events, the relative

abundance of HuD may also be regulated by post-transcrip-

tional mechanisms (see Figure 3). Northern blot analysis has

revealed the presence of two HuD transcripts of �3.7 and

4.4 kb in neurons.(114) These are thought to originate from

alternative polyadenylation or splicing to a putative down-

stream non-coding exon (exon 9).(48) Alignment of available

cDNA sequences indeed reveals the presence of alternate

30UTRs. Amongst the different Hu proteins, the 30UTRs

are unique to each transcript.(47) In contrast, specific se-

quences coding for stem-loop structures and putative ARE

within the HuD 30UTR are highly conserved between different

species.(28) In addition, the 30UTR of the Drosophila homo-

logues (ELAV and SxL) are important for the appropriate

regulation of these transcripts.(115,116) More specifically, HuB,

ELAVand SxL have been shown to autoregulate their expres-

sion through interactions with their own 30UTR.(116–118)

Recent studies have shown that ELAV expression is depen-

dent on the presence of a long UTR at its 30 end.(115) An

ELAV-binding site was in fact found within the elav mRNA

located within an alternative non-coding 30exon.(118) The

authors of this study proposed that ELAV protein binds to

elav mRNA 30UTR when protein levels reach a certain

threshold and, in this case, block further protein production.

Such a similar negative autoregulatory loop also can be

assigned to SxL protein, which can downregulate its own

expression by binding to specific regions within the

sxl 30UTR.(116) Together, these observations are coherent

with the notion that, in addition to transcriptional events

(see paragraph above), post-transcriptional mechanisms

operating through the 30UTR are also important for control-

ling HuD expression.

Post-translational regulatory mechanismsAlthough the abundance of RBP is important to ultimately

control the fate of specific transcripts, other factors such as the

RBPaffinity and function are also implicated. In a recent study,

wehaveshown thatHuDdidnot actively bind tooneof its target

transcripts until neuronal differentiation was stimulated, even

though significant levels of HuD protein were present in

undifferentiated PC12 cells and these levels did not change

upon differentiation (see Refs 42,49,62). This suggests that

additional factors affect the ability of HuD to stabilize target

transcripts. In this context, several recent studies have begun

to elucidate the pathways regulating post-translational mod-

ification of the Hu family of RNA-binding proteins including

phosphorylation andmethylation, and to determine the impact

of these modifications on their activity.(54,62,119) Notably, NGF-

induced differentiation and stress-related stimuli can indirectly

regulate HuD and HuR function, respectively, by a PKC-

mediated signaling cascade.(62,120) Recently, a series of

indirect experiments led to the suggestion that threonine

phosphorylation of the neuronal Hu proteins (HuB, HuC and

HuD) by PKCa isozyme results in the re-distribution of the

proteins and in an increased stabilization of GAP-43 mRNA,

a well-known HuD target.(54)

In addition to phosphorylation, a recent study has shown

that arginine residues in the HuR hinge region are methylated

by the methyltransferase CARM1 (coactivator-associated

arginine methyltransferase 1) in response to lipopolysacchar-

ide.(119) Arginine methylation is another common modification

of some RBP that results in modification in the pattern of

protein–protein interactions. Along those lines, it has also

recently been demonstrated in PC12 cells that HuD is

methylated on the corresponding arginine (Arg236, see Fig. 1)

by the same methyltransferase, and that methylation results

in decreased RNA-binding activity of HuD.(121) In these

studies, methylation-resistant mutant HuD increased expres-

sion of some target transcripts such as p21cip1/waf1, and

resulted in increased neuronal differentiation of PC12 cells.

Although these cell culture studies suggest that phosphoryla-

tion and methylation can regulate HuD function directly or

indirectly, it should be noted that these studies have been

performed in vitro. Whether post-translational modifications

are also occurring in vivo still remains to be determined.

Nonetheless, these studies indicate that post-translational

modifications are also likely to be important in regulating the

binding and functional activity of HuD on target transcripts.

Finally, the fate of target transcripts may also depend on

other factors that might modulate the binding activity and

specificity of HuD. These include the structure and sequence

of the target transcripts and the interaction with additional

RBPs. The transcript itself can, in many cases, modify the

function of RBP (see for review Refs 52,122). An example of

this was given above with the poly(A) tail since the presence

and length of the poly(A) tail can alter the binding of theRRM to

Review articles

BioEssays 28.8 829

the ARE.(64,67) The presence of HuD in RNP granules and the

suggested multimerization of HuD implies that interactions

with other proteins or RBP can modulate the activity of HuD

and its specificity in a manner analogous to the interactions of

transcription factors on promoter elements.(47,79,85,123) This

may explain in part the identification of numerous RBPs

interacting with a single transcript such asGAP-43, AChE and

the early-response genes c-myc and c-fos (see for review

102).(42,61,62,124)

Conclusion and perspective

To date, there is compelling evidence indicating that, through

its ability to bind and stabilize a specific subset of transcripts,

HuD plays an essential role in the development and

maintenance of neuronal phenotype. The downstream effects

of this specific activity include the stimulation of morphological

differentiation of newly committed and developing neurons.

Additionally, HuDexpression andbindingactivity have recently

been shown to increase during nerve regeneration as well as

with learning andmemory. The ability of HuD to directly control

these key molecular and cellular events indicates that HuD

may act as an important regulator of neuronal differentiation

and function. Therefore, a thorough understanding of the

mechanisms affecting the expression, activity and function of

HuD in neurons appears warranted in order to increase our

basic knowledge of the events regulating neuronal differentia-

tion, maintenance and plasticity. Ultimately, this knowledge

could prove useful for the development of novel therapeutic

strategies aimed at manipulating, pharmacologically or

through gene therapy approaches, the levels of RBP that

may be beneficial for the treatment of several neurological

diseases and conditions.

Acknowledgments

We thank members of the Jasmin laboratory for fruitful

discussions.

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