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Review

Vitamin D and alternative splicing of RNA

Rui Zhou a,b, Rene F. Chun a, Thomas S. Lisse c, Alejandro J. Garcia a, Jianzhong Xu b,John S. Adams a, Martin Hewison a,*aUCLA Orthopaedic Hospital, Department of Orthopaedic Surgery, Orthopaedic Hospital, University of California at Los Angeles, Los Angeles, CA 90095, USAbDepartment of Orthopaedics, the Orthopedic Surgery Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing 400038,ChinacMount Desert Island Biological Laboratory, 159 Old Bar Harbor Road, Salisbury Cove, ME 04672, USA

A R T I C L E I N F O

Article history:Received 1 August 2014Received in revised form 23 September 2014Accepted 26 September 2014Available online xxx

Keywords:Vitamin DRNATranscriptionSplicingHeterogenous nuclear ribonucleoprotein C

A B S T R A C T

The active form of vitamin D (1a,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects viabinding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDRbinding locations across the complete genome has significantly expanded our understanding of theactions of vitamin D and VDR on gene transcription. However, these studies have also promotedappreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear thatvitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, andmicroRNA generation to maintain normal biological functions. There is also increasing evidence thatvitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although themechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcriptionRNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroidhormones have been recognized to coordinately control gene transcription and pre-mRNA splicingthrough the recruitment of nuclear receptor co-regulators that can both control gene transcription andsplicing. The current review will discuss this concept with specific reference to vitamin D, and thepotential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with anestablished function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptionalcomplex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factorlinking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide anadditional mechanism for the fine-tuning of vitamin D-regulated target gene expression.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

ã 2014 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Journal of Steroid Biochemistry & Molecular Biology

journal homepage: www.else vie r .com/locate / j sbmb

1. Introduction

Alternative splicing provides an efficient mechanism by whichthe genome of any given organism can greatly expand its proteinrepertoire. The aim of this review is to describe our currentunderstanding of the link between alternative splicing and vitaminD. The review will initially focus on the canonical transcriptionalactions of vitamin D, but will then explore the interaction betweenvitamin D and a key component of the machinery associated withthe maintenance of splicing fidelity, heterogeneous nuclearribonucleoprotein C (hnRNPC). In this way, the review willhighlight a potentially important new mechanism for furtherexpansion of the genomic actions of vitamin D.

* Corresponding author. Tel.: +1 310 206 1625; fax: +1 310 825 5409.E-mail address: mhewison@mednet.ucla.edu (M. Hewison).

http://dx.doi.org/10.1016/j.jsbmb.2014.09.0250960-0760/ã 2014 Elsevier Ltd. All rights reserved.

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1.1. Genomic actions of the vitamin D receptor

1a,25-dihydroxyvitamin D [1,25(OH)2D], the biologically activemetabolite of vitamin D, performs the majority of its biologicalfunctions by regulating gene transcription through a nuclear high-affinity vitamin D receptor (VDR), a member of the superfamily ofnuclear receptors that bind steroid hormones and other lipophilicligands [1,2]. Upon ligand (1,25(OH)2D) binding, VDR heterodime-rization occurs with the nuclear retinoid X receptor (RXR) [3]. Theresulting VDR–RXR complex can then bind to specific DNAsequences, termed vitamin D-response elements (VDREs) withinproximal or distal promoter regions of target genes [4,5]. Afterbinding to a VDRE, a variety of VDR-interacting nuclear proteins(co-regulators) are recruited to the pre-initiation transcriptionalcomplex, which act to enhance or suppress the rate of genetranscription by the liganded VDR [6,7]. Other factors that add tothe diversity of VDR-mediated regulation of gene expression

e splicing of RNA, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.

Table 1Overview of splice variants of the CYP24A1 and CYP27B1 genes.

Gene Transcriptvariant

Organism/cell Proteincoding

Enzymaticactivity

Biological meaning Reference

CYP24A1 CYP24A1-wild type

Full length All VDR-expressingcells

Yes Yes 23- and 24-hydroxylation of 25(OH)D/1,25(OH)2D [38]

CYP24A1-SV

Deletion exons 1 and 2,insertion part of intron 2

Chick macrophage cellline, Human/kidney,placenta, skin, andmacrophages

Yes No Suppress synthesis of 1,25(OH)2D as a cytosolicdecoy for CYP24A1/CYP27B1 substrates

[39–41]

CYP24A1-SV2

Deletion exons 1 and 2 Human/colon cancercells

Unknown No Unknown

CYP24A1-SV3

Deletion exon 10 Human/colon cancercells

Unknown No Unknown

–

CYP27B1 CYP27B1-wild type

Full length Human/renal or extra-renal cells

Yes Yes 1a-Hydroxylation of 25(OH)D [38]

SV1 Contains part of intron 2,exons 6–9

Human/kidney celllines

Noncoding – Predicted function: coordinate localized synthesis of1,25(OH)2D by limiting the availability of the wildtype 1a-hydroxylase enzyme activity

[32]

SV2 Contains part of intron 2,part of exon 6, exons 7–9

Human/kidney celllines

Noncoding –

SV3 Contains part of intron 2,exon 3, part of intron 3,part of exon 8 and exon 9

Human/kidney celllines

Noncoding –

SV4 Contains part of intron 2,exon 3, intron 3, part ofexon 4 and exon 9

Human/kidney celllines

Noncoding –

SV5 Contains part of intron 2,exons 3–4 and exon 9

Human/kidney celllines

Noncoding –

SV6 Contains intron 2, exons3–5, intron 5 and part ofexon 6

Human/myelomonocytic celllines

Noncoding –

SV7 Contains part of intron 2,exons 3–5, part of exon 6and part of exon 7

Human/myelomonocytic celllines

Noncoding –

SV8 Contains part of intron 2,part of exon 4, exon 5,intron 5, exon 6 and partof exon 7

Human/myelomonocytic celllines

Noncoding –

SV9 Contains part of intron 2,part of exon 4, exons 5-6and part of exon 7

Human/myelomonocytic celllines

Noncoding –

SV10 Contains part of intron 2,part of intron 5, exon 6,and part of exon 7

Human/myelomonocytic celllines

Noncoding –

Hyd-V1 Insertion part of intron 2,deletion exons 4 and 5

Human/GBM cell lines Yes No Predicted function: reduce 1a-hydroxylase enzymeactivity; contribute to the development ofneoplasias via effects on cell-specific synthesis of1,25(OH)2D; potential as a diagnostic marker forcancer

[29–31,33,36]

Hyd-V2 Deletionexons 4 and 5 Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V3 Deletion of part of exon 8 Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V4 Insertion part of intron 2 Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V5 Insertion part of intron 5 Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V6 Insertion part of intron 1 Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V7 Insertion part of intron 1,deletion exons 4 and 5

Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V8 Insertion introns 1 and 5 Human/GBM,melanoma, cervixcarcinoma and kidneycell lines

Yes No

Hyd-V9 Insertion introns 2 and 6 Human/GBM cell lines Yes NoHyd-V10 Insertion introns 1 and 2 Human/GBM cell lines Yes NoHyd-V11 Insertion introns 1 and 3 Human/GBM cell lines Yes No

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Table 1 (Continued)

Gene Transcriptvariant

Organism/cell Proteincoding

Enzymaticactivity

Biological meaning Reference

Hyd-V12 Insertion introns 1, 3 and5

Human/GBM cell lines Yes No

Hyd-V13 Insertion introns 1, 2 and5

Human/GBM cell lines Yes No

Hyd-V14 Insertion introns 1, 3 and6, 7

Human/GBM cell lines Yes No

Hyd-V15 Insertion intron s1, 2 and3, 5

Human/GBM cell lines Yes No

Hyd-V16 Insertion introns 2 and 5,deletion exons 6–9

Human/GBM cell lines Yes No

–

Insertion intron 1 Human endometrialcancer cells/breastcancer cells

Yes No Unknown [34,35]

Deletions exons 3–5 Human endometrialcancer cells/breastcancer cells

Yes No

GBM: glioblastoma multiforme.

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include the presence of distal enhancer elements [8], as well as thepotential for ligand-independent actions of VDR [9].

As the only high-affinity receptor for 1,25(OH)2D, the VDRmediates genomic responses to 1,25(OH)2D [10]. Recent develop-ments in deep sequencing technologies for unbiased analysis ofVDR binding loci have expanded our understanding of the genome-wide actions of 1,25(OH)2D-VDR. Chromatin immunoprecipitation(ChIP) followed by DNA sequencing of the immunoprecipitatedproducts, ChIP-sequencing (ChIP-Seq), using a variety of cell typeshas shown that there are between 1000 and 13,000 VDR-specificgenomic binding sites [11,12]. The majority of these VDR bindingsites appear to be distal to the target gene transcriptional start site,being located in either intergenic regions or within introns. Thishas had a dramatic effect on our understanding of transcriptionalregulation by the 1,25(OH)2D-VDR complex. Most VDR-bindingDNA loci have yet to be validated by specific quantitative PCRanalysis of ChIP products, but, recent work by Pike and co-workershas confirmed that 1,25(OH)2D regulation of the genes for24-hydroxylase (CYP24A1) and RANK ligand (RANKL), involvesboth proximal promoter VDREs and a complex set of downstreamor upstream distal VDREs [13–15].

Genomic binding of VDR is also highly cell-specific. Tuoresmakiet al. recently conducted a re-analysis of publically available VDRChIP-seq datasets for six different cell types. These datasetsrevealed a total of 23,409 non-overlapping genomic VDR bindingsites, with a majority of these VDR loci (17,700) being unique toeach of the analyzed cell types, and only 43 binding siteswereshared by all six cell types, emphasizing the cell-specificity ofVDREs [16]. However, the underlying mechanisms that definecell-specific patterns of VDR binding remain unclear, and mayinvolve alternative accessory factors for VDR. Intriguingly morethan 60% of genomic VDR binding sites do not contain a canonicalhexameric VDRE sequence direct-repeat (DR) 3 with two viablesix-base half-elements separated by three base pairs withconsensus sequence RGGTCAnnnRGTTCA, (r = A or G), suggestingthat VDR may use alternative mechanisms to interact withgenomic DNA [16]. For example, liganded VDR may partner withcurrently undefined partner proteins or interface with otherDNA-binding transcription factors, such as pioneer factors.

These alternative binding mechanisms may explain some of thecell-specific actions of VDR as well as its repressive functions ongene transcription [11]. However, there is now strong evidencesupporting the contribution of other mechanisms that diversify theeffects of vitamin D on the transcriptome and proteome. Forexample, epigenetic modifications are known to play a key role inthe maintenance of VDR-directed gene expression and

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dysregulation of these mechanisms can lead to pathologicalconditions [17,18]. The impact of epigenetics on VDR signalinghas been well defined for chromatin remodeling via DNAmethylation and histone acetylation, with VDR co-activators andco-repressors interfacing with chromatin modifiers and remod-elers [6,19,20]. In addition, recent studies have also implicatedmicroRNAs (miRNAs) in mediating the fine-tuning of vitaminD-mediated responses [21–23]. The liganded VDR complex caneither suppress or induce miRNAs by either direct transcriptionalregulation of autonomous miRNA genesor indirect regulation ofmiRNAs via host gene promoter sequences [24]. Conversely,miRNAs may act to regulate 1,25(OH)2D synthesis, catabolism, orsignaling to form dynamic feedback mechanisms (comprehensivereview, see [24]).

The current manuscript will review another mechanism withthe potential to influence vitamin D regulated gene expression –

namely RNA splicing, and alternative splicing. In particular, thisreview will focus on the potential role of hnRNPC, a key nuclearfactor in post-transcriptional RNA-processing, as a mediator ofvitamin D receptor-directed transcription and RNA splicing.

2. Vitamin D and RNA splicing

2.1. Pre-mRNA splicing and alternative splicing overview

In humans and other complex metazoans, the vast majority ofprotein-coding genes contain several exons separated by intronsthat will not appear in the mature mRNA. Removal of introns andthe ligation of exons that contain the protein-coding open readingframe and the 50 and 30 untranslated regions (UTRs) is accom-plished by pre-mRNA splicing, a process which is facilitated by acomplex of small nuclear RNAs (snRNAs), splicing factors, andnumerous RNA-binding proteins that collectively form thespliceosome [25]. Nuclear pre-mRNA splicing entails two conse-cutive trans-esterification reactions. First, the 20-hydroxyl of anadenosine of the branch point sequence in the intron carries out anucleophilic attack on the phosphodiester bond at the 50 splicingsite (SS). This results in cleavage at this site and ligation of the50 end of the intron to the branch adenosine, forming a free 50 exonand a lariat structure. Subsequently, the phosphodiester bond atthe 30 SS is attacked by the 30-hydroxyl of the 50 exon, leading to theligation of the 50 and 30 exons and release of the lariat intron.

During splicing, the spliceosome dynamically, and in a stepwisefashion, assembles and disassembles across the pre-mRNA todirect the correct recognition and pairing of the splice sites [26].Whilst some exons are constitutively spliced, in that they are

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included in every mRNA produced from a given pre-mRNA, manyothers are alternatively spliced to generate variable forms ofmRNA from a single pre-mRNA transcript [27]. The capacityfor alternative splicing in eukaryotes has greatly enhancedtranscriptome and proteome diversity leading to a higher orderof organismal complexity but without the need for expansion ofthe genome. Importantly, alternative splicing can also be regulateddifferently according to cell type, developmental stage, orsignal-dependent patterns [25,28].

2.2. Alternative RNA splicing and the vitamin D system

Previous studies linking vitamin D and alternative splicing havefocused on the metabolism of vitamin D (Table 1). Publishedreports have described splice variant mRNA transcripts for thevitamin D-activating enzyme 25-hydroxyvitamin D-1a-hydroxy-lase (CYP27B1) [29–35]. Data from these studies have underlined apotential role for CYP27B1 splice variants in regulating localizedsynthesis of 1,25(OH)2D [32], notably in the context of tissue-specific regulation of this active from of vitamin D [31]. Otherstudies have suggested that dysregulation of CYP27B1 splicevariant expression may contribute to the development of neo-plasias via effects on cell-specific synthesis of 1,25(OH)2D[29,30,36]. The underlying mechanisms for this have yet to bedefined, as reported splice variants of CYP27B1 appear to beexpressed in very low abundance, and do not appear to encodefunctional proteins [32]. Nevertheless, knockdown of some ofthese CYP27B1 splice variants in human kidney cells using RNA-interference resulted in enhanced conversion of 25-hydroxyvita-min D (25OHD) to 1,25(OH)2D [32], suggesting a functional role forRNA splicing with respect to vitamin D metabolism. In this contextthe most informative gene for vitamin D and alternative splicingappears to be the vitamin D-catabolic enzyme 24-hydroxylase(CYP24A1), which is potently induced in cells following exposure to1,25(OH)2D [37,38].

Alternative splicing of CYP24A1 was first reported in the chickHD-11 macrophage cell line [39]. In this instance, exons 1 and 2 ofthe cyp24a1 gene are spliced out and replaced by a pseudo exoncomposing part of intron 2 [39]. Theresulting CYP24A1-splicevariant (CYP24A1-SV) generates an in-frame shift in mRNA, usingan alternative start codon that leads to a protein lacking themitochondrial-targeting sequence for CYP24A1 which is thereforemetabolically inactive. Despite this, CYP24A1-SV appears to play apivotal role in vitamin D metabolism by acting as a decoy forsubstrate 25OHD and attenuating endogenous conversion of25OHD to 1,25(OH)2D by CYP27B1 [39]. Thus, CYP24A1 may beparticularly important in cells, such as macrophages, withsignificant intracrineCYP27B1 activity. Notably CYP24A1-SVhasbeen detected in cancer cells, in particular colorectal and prostatecancer cells [40,41]. The precise impact of the CYP24A1 splicevariant in this setting has yet to be fully defined. However, incommon with its proposed role in macrophages, this may involvedecoy actions on local vitamin D metabolism by these cells. In thisway, expression of the variant protein form of CYP24A1 may play acrucial role in the efficacy of local 25OHD metabolism, with over-abundance of CYP24A1-SV acting to diminish local concentrationsof 1,25(OH)2D at the cell-specific level.

Other studies have shown that vitamin D itself may influenceCYP24A splicing. Muindi et al. reported that 1,25(OH)2D canmodulate CYP24A1 pre-mRNA splicing in prostate cancer cells in adose- and time-dependent fashion, although the mechanism forthis is unclear [40]. Another recent study showed that1,25(OH)2Dcan also regulate CYP24A1 splicing in colon cancercells [42]. In multiple colon cell lines, RNA splicing patternsregulated by 1,25(OH)2D were shown to be associated with cellularsensitivity to 1,25(OH)2D, with more significant induction of

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CYP24A1 splicing being observed in cells that were more sensitiveto 1,25(OH)2D treatment. Regulation of RNA splicing by vitamin Dalso appears to be gene selective, with vitamin D target genes otherthan CYP24A1 showing no significant variations in splicingfollowing treatment with 1,25(OH)2D [42]. The underlyingmechanism for 1,25(OH)2D-mediated variations in CYP24A1 RNAsplicing has yet to be clearly defined, and may involve multiplesignaling pathway, such as PKAactivation and c-Jun terminalprotein kinase inhibition [42]. Moreover, there is evidence that therole of 1,25(OH)2D in pre-mRNA splicing is neither confined tocancer cells nor to theCYP24A1 gene. Collectively these studieshighlight the potential importance of RNA splicing as an alternativefacet of vitamin D-mediated regulation of gene expression, and thiswill be discussed in greater detail in the remainder of the review.

2.3. Nuclear receptor co-transcriptional splicing

It is now clear that transcriptional regulation is physically andfunctionally integrated with pre-mRNA splicing [43–45]. Suchco-transcriptional splicing has been demonstrated in a number ofdifferent organisms and has been shown to play a role incoordinating both constitutive and alternative splicing [46]. Ithas been reported that by recruiting receptor co-regulators thatcan both control gene transcription and splicing, steroid hormones(in this case progesterone and estrogen) may coordinately controlgene transcription and splicing decisions leading to alternativelyspliced transcripts [47]. This mechanism is controlled in a steroidnuclear receptor (NR) and promoter-dependent manner [47]. Inparticular a subset of hormonally recruited NR co-regulatorsincluding CoAA [48], CAPERa and CAPERb [49], SKIP [50], andco-factor of BRCA 1 (COBRA1) [51], have been identified as couplingfactors involved in coordinating steroid hormone-activated genetranscription and transcript splicing. This double-duty for the NRand co-regulators, in response to a steroid hormone signal,may actto ensure that the appropriate gene product is generated in theappropriate cell type [52].

In a similar fashion, the VDR co-regulator NcoA62/SKIP canpromote RXR-VDR dependent gene transcriptional activation orrepression in a cell-specific manner [53]. However, it has also beenidentified as a non-snRNP component of splicesome complexes,supporting a potential role for NCoA62/SKIP in pre-mRNAsplicing [54,55]. Zhang et al. demonstrated that 1,25(OH)2D isable to influence target gene splicing through recruitment ofNcoA62/SKIP. In this case the co-activator function of NcoA62/SKIPin the VDR-activated transcriptional process was demonstrated inHeLa cells by showing that NcoA62/SKIP interacts with VDR, and isassociated with recruitment of VDR to CYP24A1 VDREs followingtreatment with 1,25(OH)2D [50]. However, using glutathioneS-transferase (GST)-pull down analyses and HeLa cell nuclearextracts, NcoA62/SKIP was also shown to interact with compo-nents of the splicing machinery, such as the U5 small nuclearribonucleoprotein (snRNP) [50]. Moreover, disruption ofnative NCoA62/SKIP through expression of a dominant-negativeNCoA62/SKIP blocked conventional splicing of 1,25(OH)2D-in-duced RNA transcripts from a VDRE-driven reporter minigenecassette. This resulted in a 1,25(OH)2D-dependent accumulation ofunspliced transcripts, suggesting that NcoA62/SKIP is required forcorrect splicing in VDR-activated gene expression. The mechanismby which NcoA62/SKIP affects RNA splicing has yet to be clarified,but these data suggest that NcoA62/SKIP, a common VDR co-factor,is involved in coupling VDR mediated transcription to RNA splicing[50].

There is currently no direct evidence showing that vitamin Daffects nascent mRNA alternative splicing. However, a recentproteomic study identified a number of proteins functioning assplicing factors that were induced by 1,25(OH)2D in colon cancer

e splicing of RNA, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.

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cells [56]. As detailed above, VDR-interacting proteins withtraditional roles as VDR transcriptional co-activators or co-repressors, may also be involved in the regulation of alternativesplicing [57]. The remainder of this review will focus on analternative view of this concept, in which a nuclear factor with anestablished function in RNA splicing, hnRNPC, has also been shownto be involved in the VDR transcriptional complex as aVDRE-binding protein [58].

3. HnRNPC1/C2, RNA splicing, and VDR-mediated transcription

The protein complex known as hnRNPC is a member of asubfamily of hnRNPs that act as RNA binding proteins bycomplexing with heterogeneous nuclear RNA. Transcript variantsof the hnRNPC gene encode two major isoforms (C1 and C2) thatform an hnRNPC1/C2 heterotetramer consisting of 3 hnRNPC1 and1 hnRNPC2 subunits [59,60]. Although hnRNPC1/C2 was identifiedover 30 years ago as a core component of 40S hnRNP particles,whichform on all nascent RNA transcripts [61], conflicting reportsstill exist in regard to the sequence specificity and mode ofhnRNPC1/C2 binding to RNA [62–65]. A recent study using in vivocrosslinking and immunoprecipitation followed by deep sequenc-ing confirmed that hnRNPC1/C2 is predominantly positioned onpre-mRNAs via sequence-specific binding to neighboring uridinetracts of its RNA recognition motif (RRM) domains, with a definedspacing of 165 and 300 nucleotides [66]. In this study it washypothesized that hnRNPC1/C2 act as an ‘RNA nucleosome’ thatincorporates long regions of nuclear pre-mRNA [66]. This isconsistent with the several previously reported functions ofhnRNPC1/C2 in post-transcriptional RNA processing includingRNA packaging [67,68], constitutive and alternative splicing[69–72] and RNA export [73].

3.1. HnRNPC1/C2 and RNA splicing

An essential role for hnRNPC1/C2 in pre-mRNA splicing wasidentified over 20 years ago [69,70]. However, more recent studieshave described a versatile function for hnRNPC1/C2 in alternativesplicing. Knockdown of hnRNPC1/C2 by small interfering RNAs(siRNAs) promoted exon skipping or the use of more internal30 -splice sites, suggesting that hnRNPC1/C2 acts as a splicingenhancer by enforcing exon inclusion in a cell type-dependentmanner [71]. Another study described an opposite role forhnRNPC1/C2 in regulating alternative pre-mRNA splicing topromote exon exclusion [74]. In this latter case cooperative

Fig.1. DNA and RNA binding functions of hnRNPC1/C2 and the action of 1,25(OH)2D. Scheelement-binding protein (VDRE-BP) in the absence of liganded (1,25(OH)2D-bound) vitamfrom the VDRE; (2) a component of the RNA spliceosome, facilitating either exon excluC2 function are linked.

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interaction of hnRNPC1/C2 and human antigen R (HuR), an RNAbinding protein which has been previously identified as analternative splicing regulator in mammalian cells [74], promotedFas cell surface death receptor (Fas) gene exon 6 skipping throughbinding to the exon splicing silencer (ESS) URE6. This, in turn,inhibited molecular events leading to exon definition, andsubsequent exon skipping [72].

More recent approaches utilizing newly developed techniquessuch as individual-nucleotide resolution UV cross-linking andimmunoprecipitation (iCLIP)[75], have been able to determinemore precise locations of hnRNPC1/C2 binding to nascent tran-scripts and provide insights into the mechanism of hnRNPC1/C2regulated alternative splicing [66,76]. Using iCLIP followed by deepsequencing, Konig et al. characterized a precise transcriptome-wide binding pattern of hnRNPC1/C2 at single-nucleotide resolu-tion. These studies determined that hnRNPC1/C2 can promoteeither exon exclusion or inclusion, depending on the exact bindinglocation of hnRNPC1/C2 on the nascent mRNA. Alternative exonswere skipped by direct binding of the exon to theRNPC1/C2 tetramer, while binding of the hnRNPC1/C2 tetramerto the preceding intron enhanced the inclusion of alternativeexons [66]. Another recent study by the same team found thathnRNPC1/C2 directly competes with the core splicing factorU2 auxillary factor 65 (U2AF65), with this mechanism acting toprevent a process known as Alu exonization [76]. Sequences ofDNA known as Alu elements are amongst the most transposable ofDNA elements in the human genome. However, Alu elements canintroduce cryptic splice sites which, if used, lead to Aluexonization. There have been increasing numbers of reports ofAlu exonization-associated disorders and the mechanisms thatprevent spurious exonization of Alu elements are currentlyunknown [77]. In this setting hnRNPC1/C2 has been identifiedas a potential mediator of this protection against spuriousexonization [76].

3.2. HnRNPC1/C2 and vitamin D resistance

Despite their classical interaction with chromatin-associatedsingle-stranded RNA, several studies have shown thathnRNPC1/C2 is a pluripotent protein complex with the potentialfor alternative modes of action [58,78–80]. In particular, previousstudies from our group have demonstrated a role for hnRNPC1/C2 as an additional component of the VDR transcription complex,in which hnRNPC1/C2 functions as a trans-regulatory factor viainteraction with double-stranded DNA cis-elements[79,81,82].

matic representation of the ability of hnRNPC1/C2 to act as: (1) a vitamin D-responsein D receptor (VDR). In the presence of VDR-1,25(OH)2D, hnRNPC1/C2 is displaced

sion or exon-inclusion. As yet it is unclear whether these two facets of hnRNPC1/

e splicing of RNA, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.

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A role for hnRNPC1/C2 in vitamin D-mediated signaling wasfirst reported following an outbreak of rachitic bone disease in theNew World Primate (NWP) emperor tamarin (Saguinas imperator)colony at the Los Angeles Zoo [83]. Analysis of epidermal cells fromthese animal showed that the NWPs were protected againstpotential adverse effects of sustained exposure to high circulatinglevels of 1,25(OH)2D by elevated cellular expression of a proteinthat binds to VDR target gene promoter VDREs [79,81]. Overabun-dance of this so-called “VDRE-binding protein” (VDRE-BP) appearsto attenuate vitamin D receptor-mediated gene expression byblocking the binding of 1,25(OH)2D-liganded VDR to target genepromoter VDREs. This occurs via direct binding of the VDRE-BP tothe VDRE DNA half-sites, and has been proposed as the underlyingcause of 1,25(OH)2D-insensitivity in NWPs [81,84]. Subsequentstudies showed that a VDRE-BP was identical to hnRNPC1/C2 thatwas over-expressed in a human subject with a form of hereditaryvitamin D resistant rickets (HVDRR) [79,82,85]. In this case, ChIPanalyses confirmed the ability of hnRNPC1/C2 to compete in transwith VDR for occupation of the VDRE in human cells. In normal 1,25(OH)2D responsive human cells, the hnRNPC1/C2 occupying theCYP24A1 VDRE at baseline was then displaced from the promoterVDRE by the 1,25(OH)2D liganded VDR–RXR complex [79]. Thisreciprocal relationship between VDR and hnRNPC1/C2 occurs in atime-dependent cyclical fashion following exposure to 1,25(OH)2D, suggesting that hnRNPC1/C2 may be a key determinantof the temporal patterns of VDRE occupancy [79].In hnRNPC1/C2 over-expressing fibroblasts and B cells from the HVDRR patient,the reciprocal association of VDR and hnRNPC1/C2 with the VDREwas distorted relative to control vitamin D-responsive cells [79]. Ina similar fashion, overexpression of hnRNPC1/C2 in vitro has beenshown to disrupt the relationship between VDR and hnRNPC1/2,leading to suppression of 1,25(OH)2D-induced gene expression[79]. These data suggest that vitamin D resistance in the case of theHVDRR patient was similar to that previously described for NWPs[58,79].

3.3. hnRNPC1/C2 – a link between VDR-mediated transcription andalternative splicing?

Although there is evidence for effects of 1,25(OH)2D on pre-mRNA splicing, the mechanism for this is unclear. Because of itscapacity to interact with nascent pre-mRNA as well as double-strand DNA, we hypothesize that hnRNPC1/C2 may play a key rolein linking 1,25(OH)2D-induced transcription with RNA splicing,thereby providing an additional mechanism for fine-tuning of geneexpression (see Fig. 1). This hypothesis is supported by theincreasing evidence for functional integration between transcrip-tion and splicing machinery in mammalian cells [86]. This dual rolefor hnRNPC1/C2 may help to ensure more precise regulation ofgene expression in response to vitamin D, in a manner that is moreefficient for cellular energy. It is well recognized that thebiosynthetic and metabolic energy cost of RNA splicing andthe surveillance required to eliminate imprecisely spliced mRNAsis very high, as is the potential harm from defects in theseprocesses [25].

A key question to be answered in future studies is whetherthe hnRNPC1/C2 displaced from VDRE by liganded VDR isinvolved in facilitating RNA splicing. If this is the case, thenanother question arises as to how hnRNPC1/C2 migrates fromgene promoter regions to a specific RNA splice site to direct pre-mRNA splicing or alternative splicing. Recent studies have shownthat a ‘mediator complex’ may function to “hand over” a splicingregulator, heterogeneous nuclear ribonucleoprotein L (hnRNPL),from gene promoters to the elongating RNA polymerase IIcomplex, thereby mediating specific splice site selection duringco-transcription splicing [87]. Moreover, as outlined above, VDR

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co-activators or co-repressors may also function as splicingfactors and, as such,these may also act as coupling factors linkingtranscription and RNA splicing responses to vitamin D. Furtheranalysis of vitamin D signaling, hnRNPC expression andalternative splicing of RNA will broaden our perspective onhow vitamin D is able to influence gene expression duringnormal physiology. However, alternative splicing may also be acontributor to human disease, and it in future studies it will alsobe important to assess the impact of vitamin D status on hnRNPCfunction and normal RNA splicing.

Acknowledgements

This work was supported by NIH grants AR037399 andAR063910, and by a scholarship from the China ScholarshipCouncil (CSC).

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