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Vol. 17, Issue 12, 5105-5114, December 2006

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A Nuclear Function of Hu Proteins as Neuron-specific Alternative RNA Processing Regulators

Hui Zhu^*, Robert A. Hasman^*, Victoria A. Barron^*, Guangbin Luo^*,, and Hua Lou^*,,

*Department of Genetics, Case Comprehensive Cancer Center, and Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106

Submitted February 3, 2006; Revised September 8, 2006; Accepted September 29, 2006
Monitoring Editor: Peter Walter

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recent advances in genome-wide analysis of alternative splicing indicate that extensive alternative RNA processing is associated with many proteins that play important roles in the nervous system. Although differential splicing and polyadenylation make significant contributions to the complexity of the nervous system, our understanding of the regulatory mechanisms underlying the neuron-specific pathways is very limited. Mammalian neuron-specific embryonic lethal abnormal visual-like Hu proteins (HuB, HuC, and HuD) are a family of RNA-binding proteins implicated in neuronal differentiation and maintenance. It has been established that Hu proteins increase expression of proteins associated with neuronal function by up-regulating mRNA stability and/or translation in the cytoplasm. We report here a novel function of these proteins as RNA processing regulators in the nucleus. We further elucidate the underlying mechanism of this regulation. We show that in neuron-like cells, Hu proteins block the activity of TIA-1/TIAR, two previously identified, ubiquitously expressed proteins that promote the nonneuronal pathway of calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA processing. These studies define not only the first neuron-specific regulator of the calcitonin/CGRP system but also the first nuclear function of Hu proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Alternative RNA processing is one of the major mechanisms that expand the functional proteome from a limited genome size. Recent genome-wide bioinformatic analyses have demonstrated that the majority of human pre-mRNAs undergo alternative RNA processing (Modrek and Lee, 2002). A global survey of alternative splicing by using exon–junction microarrays indicated that at least 74% of multi-exonic human pre-mRNAs are alternatively spliced (Johnson et al., 2003). The most extensive alternative splicing occurs in brain tissues (Xu et al., 2002). It is not uncommon for hundreds of different isoforms to be generated from a single gene through alternative splicing in the mammalian nervous system. The genes that undergo extensive alternative splicing include those involved in cell-cell interactions, such as cell surface receptor molecules and various ion channels (Grabowski and Black, 2001; Black and Grabowski, 2003; Lipscombe, 2005). Significant advances have been made in dissecting the functional differences between different isoforms. As a result, it was postulated that alternative splicing plays a key role in supporting the complex functions of the nervous system (Grabowski and Black, 2001; Black and Grabowski, 2003; Lipscombe, 2005).

Although recent studies have provided exciting insight into the distinct functions played by individual alternatively spliced protein isoforms during neuronal development, understanding of the regulatory mechanisms that control neuron-specific alternative splicing remains very limited. Despite the extensive nature of alternative splicing in the nervous system, only a small number of factors involved in regulation of alternative splicing in the nervous system of mammals have been identified and even fewer characterized (Black and Grabowski, 2003). These factors include classical RRM-containing proteins such as nPTB/brPTB, NAPOR/CUGBP2, and Fox-1/Fox-2; KH-type proteins such as Nova; and the STAR/GRG family proteins such as QK1 (Ashiya and Grabowski, 1997; Zhang et al., 1999; Jensen et al., 2000; Markovtsov et al., 2000; Polydorides et al., 2000; Wu et al., 2002; Zhang et al., 2002; Dredge and Darnell, 2003; Ule et al., 2003; Underwood et al., 2005). All of these factors function by binding to their cognate target sequences on the pre-mRNA molecules and by modulating splicing of neuron-specifically processed exons either positively or negatively. In most cases, it is not clear how these factors interact with the basic splicing machinery to modulate splicing; however, one recent report demonstrated elegantly that the PTB protein blocks entry of U2AF into the presplicesomal E complex, thereby suppressing inclusion of the neuron-specific c-src N1 exon in nonneuronal cells; suppression is relieved in neuronal cells by the replacement of the ubiquitous form of PTB with a different form of PTB, nPTB/brPTB (Sharma et al., 2005). A clue to how alternative splicing regulates brain-specific functions came from a study by Ule and colleagues, in which they provided compelling evidence to support the existence of a multi-tiered network regulated by Nova. They demonstrated that, in a coordinated manner, Nova regulates the exon content of RNAs encoding a large number of proteins that interact in the synapse, which accounts for 7% of the brain-specific alternative splicing in the neocortex (Ule et al., 2005).

Mammalian Hu proteins are a group of RNA-binding proteins consisting of four family members: HuA (HuR in human), HuB (HelN1 in human), HuC, and HuD. Similar to Nova1 and Nova2, which are autoimmune targets in paraneoplastic opsoclonus myoclonus ataxia (Buckanovich et al., 1996), HuD was originally cloned by screening a cerebellar expression library by using antisera from patients with paraneoplastic encephalomyelitis, one form of paraneoplatic syndrome in which Hu proteins are the autoimmune antigens (Szabo et al., 1991). All three neuron-specific members of the Hu family, HuB, HuC, and HuD, were shown to be autoimmune antigens of the Hu syndrome (Posner and Dalmau, 1997). Several in vivo and in vitro experiments indicate an important role of neuron-specific Hu proteins in neuronal differentiation (Wakamatsu and Weston, 1997; Akamatsu et al., 1999, 2005; Anderson et al., 2000). At the molecular level, all members of the Hu protein family, including HuA (HuR) have been shown to play important roles in the cytoplasm. They interact with AU-rich elements (AREs) in 3'-untranslated regions (UTRs) to regulate mRNA stability (Jain et al., 1997; Myer et al., 1997; Anderson et al., 2000); the human HuB and HuD proteins have also been shown to modulate translation (Antic et al., 1999; Kullmann et al., 2002).

Despite evidence of nuclear-cytoplasmic shuttling (Fan and Steitz, 1998; Burry and Smith, 2006) and the predominant nuclear localization (Okano and Darnell, 1997), none of the Hu proteins have been reported to have a nuclear function. However, in Drosophila, the Hu protein homologue, the embryonic lethal abnormal visual (ELAV) protein, has been shown to have a very important function in the nucleus. ELAV regulates alternative pre-mRNA processing in neurons (Lisbin et al., 2001; Soller and White, 2003, 2005). Although the idea that Hu proteins may regulate alternative splicing in mammalian neurons seemed extremely promising, it remains a mystery whether Hu proteins can function as splicing regulators, because no target sequence of Hu proteins as alternative splicing regulators has been identified. In this report, we describe our finding of the first such target of Hu proteins, the human calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA.

Calcitonin/CGRP pre-mRNA represents one of many RNA transcripts that undergo neuron-specific regulation of alternative RNA processing. In a subset of neurons, the six exon-containing calcitonin/CGRP pre-mRNA is processed to skip the alternative 3'-terminal exon 4, leading to the production of the neurotransmitter CGRP, in contrast to thyroid C cells where this exon is included and exons 5 and 6 are skipped leading to the production of the peptide hormone calcitonin (Figure 1A) (Rosenfeld et al., 1982). Previous studies demonstrated that the rate-limiting step for processing the calcitonin/CGRP pre-mRNA is the decision about whether to include or exclude the nonneuronal exon 4, which contains suboptimal RNA processing signals (Bovenberg et al., 1986; Adema et al., 1990). An enhancer element located downstream of exon 4 was demonstrated to promote inclusion of the exon partly through enhancing polyadenylation (Lou et al., 1996). Although it was speculated that neuron-specific factors exist to regulate the CGRP-specific pathway (Leff et al., 1987; Roesser et al., 1993), previous studies have only identified a number of factors that affect the nonneuronal pathway (Lou and Gagel, 1999; Tran et al., 2003; Tran and Roesser, 2003; Zhu et al., 2003; Roesser, 2004).

View larger version (50K): [in this window] [in a new window]   Figure 1. Alternative pre-mRNA processing of the calcitonin/CGRP gene. (A) Schematic diagram of the calcitonin/CGRP gene and its alternative RNA processing in thyroid and neuronal cells. (B) Diagram showing the calcitonin/CGRP reporter gene and its cell-specific RNA processing patterns. (C) Left, RT-PCR assay of total RNA from HeLa or CA77 (rat medullary thyroid carcinoma) cells transfected with the diagramed calcitonin/CGRP reporter gene. Amplification bands resulting from exon 4 inclusion or exclusion are indicated. Right, a picture of CA77 cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids
The human calcitonin/CGRP reporter constructs used in transfection experiments consist of calcitonin/CGRP exons 4–6 fused to a heterologous first exon from adenovirus (Lou et al., 1995). The reporter construct that contains mutations at the U-tract sequences (UUUUUUAUUUU to GUGUUGAUGGU) and plasmids used to generate in vitro transcribed RNA substrates for UV cross-linking and gel-shift assays were described previously (Zhu et al., 2003). The calcitonin/CGRP reporter construct containing a Rev-responsive element (RRE) sequence was generated by insertion of an XhoI restriction enzyme recognition sequence upstream of the U-tract through polymerase chain reaction (PCR)-directed mutagenesis followed by ligation of two oligonucleotides complimentary to each other at the XhoI site (sense oligonucleotide, 5'-TCGAGTGGGCGCAGCGTCAATGACGCTGACGGTACAC; and antisense oligonucleotide, 5'-TCGAGTGTACCGTCAGCGTCATTGACGCTGCGCCCAC).

To generate cDNA sequences of the mouse HuB and HuC, reverse transcription (RT)-PCR was carried out using RNA isolated from the mouse F9 cells and mHuB- or mHuC-specific oligonucleotides (5'-GCGGATCCAATGGAAACACAACTGTCTAA and 5'-GCGAATTCTTAGGCTTTGTGCGTTTTGTT for mHuB and 5'-GCGGATCCAATGGTCACTCAGATACTGGG and 5'-GCGAATTCTCAGGCCTTGTGCTGCTTGC for mHuC). The PCR products were digested with BamHI and EcoRI and cloned into the BamHI and EcoRI sites in the pGEX-2TK (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) vector for glutathione S-transferase (GST) recombinant proteins production or the BamHI-EcoRI sites in the pcDNA3.1HisB (Invitrogen, Carlsbad, CA) vector for mammalian cell transfection. Truncated mHuC constructs were generated by similar PCR-directed cloning. mHuC RRM12 and mHuC RRM3 were created using oligonucleotide pairs 5'-GCGGATCCAATGGAAACACAACTGTCTAA and 5'-GAATTCGTTTGCGAACTTGACCGT, and 5'-GCGGATCCAAAGACAGGGCAGGCCCTGCT and 5'-GCGAATTCTCAGGCCTTGTGCTGCTTGC, respectively. The TIA-1-Rev and HuC-Rev plasmids were constructed by PCR amplifying TIA-1 or HuC and inserting the PCR product into the SphI and BglII sites of the PC12-Rev vector (a gift from Dr. Christine Herrmann, Baylor College of Medicine, Houston, TX). All of these plasmids were verified by DNA sequencing.

Cell Culture, P19 Differentiation, and Cell Transfection
HeLa, Chinese hamster ovary (CHO), F9, PC12 TT, SK-N-SH, and P19 cell lines were maintained according to instructions from American Type Culture Collection (Manassas, VA). CA77 cells, a cell line derived from rat medullary thyroid carcinoma (a gift from Drs. Alison Hall at Case Western Reserve University, Cleveland, OH, and Andrew Russo, University of Iowa, Iowa City, IA) were cultured in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% Pen/Strep (Invitrogen). For differentiation of P19 cells, cells were aggregated in bacterial culture dishes and either untreated or treated with 0.3 µM all-trans retinoid acid (Sigma-Aldrich, St. Louis, MO) or 1% dimethyl sulfoxide (DMSO) for 4 d and then plated in tissue culture dishes. Transfection of differentiated P19 cells was carried out 48 h after the cells were transferred to tissue culture dishes by using Lipofectamine (Invitrogen), and cells were collected 4 d after transfection for analysis. HeLa cells were transfected as described previously (Zhu et al., 2003). Transfection of CA77 cells was carried out essentially the same as HeLa cells except that Lipofectamine 2000 (Invitrogen) was used, and cells were grown for 72 h before and after transfection. Cotransfections used 1 µg of the calcitonin/CGRP reporter plasmid and either 0.2–0.4 µg of TIA-1, Rev, TIA-1-Rev or HuC-Rev plasmids, or 1–2 µg of mHuC RRM12 or mHuC RRM3 plasmids.

RNA and Protein Analysis
Procedures for total RNA and protein isolation and RT-PCR analysis were described previously (Zhu et al., 2003). Use of low cycle (18–22) PCR permitted determination of the relative abundance of individual RNA species. Quantification of exon inclusion was determined using a PhosphorImager (GE Healthcare). The results shown are representative of at least three independent transfections for each experiment. The effect of TIA-1, Rev, TIA-1-Rev, HuC-Rev, mHuC RRM12, or mHuC RRM3 on RNA processing of the reporter pre-mRNA was calculated as percentage of the calcitonin exon inclusion [calcitonin exon inclusion/(calcitonin exon inclusion + CGRP exon inclusion)]. Western blot analysis using the proteins isolated from the untransfected or transfected cells was carried out with anti-Xpress antibody (Invitrogen) or anti-TIA-1/TIAR antibody (3E6) (Zhu et al., 2003) and anti-Hu sera (a gift from Dr. Jerome Posner, Sloan Kettering Cancer Center, New York, NY). Because it is difficult to detect overexpressed proteins in CA77 cells due to the extremely low transfection efficiency, protein expression of Rev, TIA-1-Rev, mHuC-Rev, mHuC RRM12, and mHuC RRM3 was verified using HeLa cells.

In Vitro Assays
UV cross-linking reactions were carried out as described previously (Zhu et al., 2003). Cross-linked polypeptides were immunoprecipitated using monoclonal antibodies against TIA-1 and TIAR (3E6) or anti-Hu sera. Gel-shift assays were performed using recombinant GST-TIAR or His-HuD prepared from bacteria and in vitro-transcribed RNA substrates. The reactions were described previously (Zhu et al., 2003).

Immunoprecipitation of CA77 Nuclear Extract and RT-PCR Analysis
Eight hundred micrograms of CA77 cell nuclear extract proteins was immunoprecitated with anti-Hu sera in NET supplemented with RNase Out (Invitrogen). The pellet was washed five times in NET (150 mM NaCl, 50 mM Tris, pH 7.5, and 0.05% NP-40), treated with proteinase K, and extracted with phenol/chloroform, followed by RNA precipitation. RT-PCR was carried out as described previously (Zhu et al., 2003). Oligonucleotides used for RT-PCR were TTAGCCCCGGAGGTTAAGCA and TTGATGCCACCTCTGAGCCT for calcitonin/CGRP (both in intron 4) and ACCTCCAAACTGAAGAGC and TGGCAGGTTTCTCCAGGCGGC for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (in intron 3 and exon 5, respectively).

Nuclear Extract Preparation from HeLa and CA77 Cells
HeLa cell nuclear extracts were prepared using S3 suspension culture and standard techniques (Zhu et al., 2003). To make nuclear extracts from CA77 cells, 100 100-mm dishes of monolayer of CA77 cells were collected and used following a standard procedure (Zhu et al., 2003).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Correlation between Hu Protein Expression and CGRP-specific Processing
In mammals, Hu proteins comprise a family of RNA-binding proteins that contains four closely related members: HuA (HuR in human), HuB (HelN1 in human), HuC, and HuD. Three of these proteins, HuB, HuC, and HuD, are normally exclusively expressed in all neurons of the central and peripheral nervous systems with the exception of HuB, which is also expressed in germ cells (Okano and Darnell, 1997). Although distinct combinations of the individual Hu proteins do exist for individual neurons, every neuron has at least one Hu protein (Okano and Darnell, 1997). CGRP is naturally produced in a subset of neurons, including hippocampal neurons, trigeminal ganglion, dorsal root ganglion, and spinal cord (Rosenfeld et al., 1992). Thus, a correlation between Hu protein expression and CGRP production is obvious in vivo.

Several cell lines have been used in previous studies that recapitulate either the calcitonin- or CGRP-specific RNA processing pathway. In our studies, we use HeLa cells to mimic the calcitonin pathway, and CA77 cells derived from rat medullary thyroid carcinoma (MTC) to mimic the CGRP pathway. HeLa cells do not express the calcitonin/CGRP gene endogenously. However, when transfected with a calcitonin/CGRP reporter construct that contains a fragment of the human calcitonin/CGRP gene extending from the middle of intron 3 to the 3' end of the gene fused with a first exon and half of an intron from the major late transcription unit of adenovirus, these cells process the reporter pre-mRNA in the calcitonin-specific pathway (Figure 1, B and C, lane 1). To examine CGRP-specific processing, we chose CA77 cells in our current study. These cells provide an optimal system for our purposes because although the line is derived from a rat MTC, medullary thyroid carcinoma, the CA77 cells have a neuronal phenotype characterized by neurites and neuronal antigens (Russo et al., 1992) (Figure 1C, right). Most relevant to us is the endogenous expression of the calcitonin/CGRP gene and the switch from 95% calcitonin to as high as 80–90% CGRP mRNA production (Russo et al., 1992). When CA77 cells are transfected with the reporter construct shown in Figure 1B, processing of the reporter pre-mRNA closely mimics that of the endogenous calcitonin/CGRP, with CGRP-specific processing as the predominant pathway (Figure 1C, lane 2) (Russo et al., 1992).

To examine the expression level of Hu family members, proteins in the whole cell lysate isolated from eight cell lines were separated on SDS-PAGE and probed with anti-Hu sera derived from patients who suffer from a paraneoplastic syndrome in which Hu proteins are autoimmune antigens. The anti-Hu sera can detect all three neuron-specific Hu proteins (HuB, HuC, and HuD) but not the ubiquitously expressed HuA (HuR) in a Western blot analysis. Strong signals were observed in F9, PC12, TT, CA77, mouse embryonic stem (ES) cells, and SK-N-SH cells, where the CGRP pathway is preferred based on published and unpublished work (Bennett and Amara, 1992; Russo et al., 1992; our unpublished data). In contrast, no Hu signals were detected in the HeLa and CHO cells, where the calcitonin pathway is preferred (Figure 2A) (Lou et al., 1996, 1998). Expression of a group of generally expressed splicing regulators, TIA-1 and TIAR, did not show significant differences among these cell lines (Figure 2A). In an attempt to further correlate Hu protein expression level and CGRP production, we isolated total RNA from four cell lines that endogenously express both Hu proteins and the calcitonin/CGRP gene and carried out RT-PCR analysis. It is very clear that the highest CGRP expression was observed in the two cell lines that express very high levels of Hu proteins, F9 and mouse ES cells. Note that only one-sixth of the total protein lysate was loaded for these two cell lines in the blot probed with anti-Hu sera. In CA77 and TT cells, where the Hu protein level is much lower, a higher calcitonin expression level was observed (Figure 1A).

View larger version (37K): [in this window] [in a new window]   Figure 2. Expression of Hu proteins correlates with the CGRP processing pathway. (A) Hu proteins are highly expressed in cells that undergo CGRP-specific RNA processing. Western blot analysis was carried out with human anti-Hu serum or anti-TIA-1/R antibody. Splicing pathways were determined by RT-PCR of total RNA isolated from these cells with (for cells that do not express the gene endogenously) or without transfection of the calcitonin/CGRP reporter gene. HeLa and CHO cells have been used as cell models for the calcitonin pathway, whereas F9 (mouse teratocarcinoma) and PC12 (rat pheochromocytoma) cells have been used as cell models for the CGRP pathway. CA77 (rat) and TT (human) cells are derived from medullary thyroid carcinomas, and SK-N-SH is a human neuroblastoma cell line. One-sixth of total proteins was loaded in the lanes F9 and mouse ES in the top panel. RT-PCR analysis of RNA isolated from cells that express the calcitonin/CGRP gene was shown. The calcitonin product is 308 nucleotides for rat and mouse and 276 nucleotides for human. The CGRP product is 284 nucleotides for rat and mouse and 237 nucleotides for human. (B) Correlation between expression of neuron-specific Hu proteins (HuB, HuC, and HuD) and the neuronal RNA processing phenotype. A calcitonin/CGRP reporter gene was transfected with untreated RA-induced or DMSO-induced mouse P19 cells. Total RNA was isolated from the transfected cells, and semiquantitative RT-PCR was carried out using primers specific for mouse HuA, HuB, HuC, HuD, GAPDH, or the calcitonin/CGRP reporter gene. Amplification bands resulting from exon 4 inclusion (nonneuronal) or exclusion (neuronal) are indicated. The percentage of inclusion of exon 4 is indicated below each lane.

Hu Proteins and TIA-1/TIAR Compete for Binding to the Same U-rich Sequence Located in a Previously Characterized Intronic Enhancer
Hu proteins have been shown to have strong affinity for U-rich sequences (Chung et al., 1996). They bind to ARE sequences in the 3'-UTRs of many mRNAs to regulate mRNA stability (Brennan and Steitz, 2001; Keene, 2001; Perrone-Bizzozero and Bolognani, 2002). Previously, we identified a U-tract sequence located in an intronic element downstream of the calcitonin exon 4 and showed that it plays an important role in inclusion of this exon in nonneuronal cells (Lou et al., 1995). Furthermore, we demonstrated that TIA-1/TIAR proteins bind to the U-tract sequence and promote inclusion of the exon (Zhu et al., 2003). We hypothesized that in neurons, Hu proteins bind to the same sequence and block access by the TIA-1/TIAR proteins that are also present in neurons (Figure 2A). To determine whether the U-tract sequence is a Hu protein-binding target, UV cross-linking/immunoprecipitation (IP) was carried out in CA77 nuclear extract by using Hu anti-sera derived from patients with Hu syndrome. Hu proteins are abundantly expressed in these cells (Figure 2A). As expected, we detected a strong signal indicative of Hu proteins binding to an RNA substrate containing the U-rich sequence, but not to an RNA containing the mutated sequence (Figure 3, A and B). Note that the same mutations were also shown to abolish the binding of TIA-1/TIAR proteins (Zhu et al., 2003). The gel mobility shift assay using the same wild-type and mutant RNA substrates showed a result consistent with the UV cross-linking/IP experiment (Figure 3C).

View larger version (32K): [in this window] [in a new window]   Figure 3. Hu proteins interact with the U-tract in the intronic element. (A) RNAs used in the UV cross-linking/IP and RNA gel mobility shift assay. Wild-type and mutated U-tract sequences are indicated. (B) UV cross-linking/IP assay. The 32P-labeled in vitro transcribed RNAs were UV cross-linked in CA77 cell nuclear extract and immunoprecipitated with antibodies specific to Hu proteins. (C) RNA gel mobility shift assay. The 32P-labeled in vitro transcribed RNAs were incubated with 0, 1, 2, 10, and 50 ng of GST-HuB.

View larger version (46K): [in this window] [in a new window]   Figure 4. Hu proteins compete with TIAR for binding at the U-tract sequence. (A) Gel mobility shift assay. The 32P-labeled in vitro transcribed RNAs indicated in Figure 3A were incubated with GST-TIAR and His-HuD. Lane 1, no protein added; lanes 2–5, 0.1 µg of His-HuD and 0, 0.02, 0.2, and 2 µg of GST-TIAR were added; and lanes 6–9, 0.1 µg of GST-TIAR and 0, 0.02, 0.2, and 2 µg of His-HuD were added. (B) UV cross-linking/IP assay. Top, 32P-labeled in vitro-transcribed RNAs was UV cross-linked in CA77 cell nuclear extract in the absence (lane 1) or presence of 0.2, 1, and 5 µg of GST-TIAR (lanes 2–4) and immunoprecipitated with antibodies specific to Hu proteins (anti-Hu sera) and TIA-1/TIAR (3E6). Bottom, 32P-labeled in vitro-transcribed RNAs were UV cross-linked in HeLa cell nuclear extract in the absence (lane 1) or presence of 0.1, 0.5, and 2.5 µg of GST-mHuC (lanes 2–3) and immunoprecipitated with antibodies specific to Hu proteins (anti-Hu sera) and TIA-1/TIAR (3E6). (C) Reduced binding of TIA-1/TIAR to the U-tract sequence in CA77 cells. Left, Western blot analysis using same amounts of nuclear proteins from HeLa and CA77 cells and anti-TIA-1/TIAR antibody. Right, UV cross-linking/IP assay using 32P-labeled RNA substrate indicated in Figure 3A, same nuclear protein amounts of HeLa and CA77 cell nuclear extracts, and anti-TIA-1/TIAR antibody.

Tethering of TIA-1 Protein in the Vicinity of the U-Tract in CA77 Cells Promotes Exon 4 Inclusion
Our previous experiments using HeLa cells show that TIA-1/TIAR proteins are required for exon 4 inclusion (Zhu et al., 2003). Because interaction of TIA-1/TIAR with the U-tract is dramatically reduced in CA77 cells, we reasoned that if we could increase binding of this protein, we might be able to increase exon 4 inclusion in these cells. We first tried to overexpress TIA-1 or TIAR in CA77 cells, but we observed no increase in exon 4 inclusion (our unpublished data). This result was not unexpected because binding of TIA-1/TIAR to the U-tract may remain low in CA77 cells even when the overall protein level is higher due to the presence of endogenous Hu proteins. Next, we took advantage of a well-established fusion protein approach to target TIA-1 to the vicinity of the U-tract sequence. This approach has been successfully used to bring protein factors to RNA targets (Tiley et al., 1992) and is similar to the MS2-MS2 coat protein fusion approach. We introduced a human immunodeficiency virus RRE immediately upstream of the U-tract of the calcitonin/CGRP reporter gene (Figure 5A) and brought TIA-1 to the sequence through a TIA-1-Rev fusion protein. The introduced RRE sequence contains a minimal, 29-nucleotide single stem-loop structure shown to have strong binding for Rev (Tiley et al., 1992). The resulting reporter construct behaved like the parental construct in that its pre-mRNA was processed to predominantly skip exon 4 (Figure 5B, lane 1, compare to lane 2 in Figure 1C). However, when a TIA-1-Rev fusion protein construct was cotransfected with the RRE-containing reporter gene, exon 4 inclusion was increased by 2- to 2.5-fold, whereas cotranfection of Rev alone construct did not significantly affect RNA processing of this reporter gene (Figure 5B, lanes 2–5). As expected, cotransfection of TIA-1 with the RRE-containing reporter did not change the splicing phenotype (our unpublished data). These experiments establish that reduced binding of TIA-1/TIAR at the U-tract sequence is responsible for the low level of exon 4 inclusion in CA77 cells.

View larger version (34K): [in this window] [in a new window]   Figure 5. Targeted binding of TIA-1 to the U-tract region increased exon 4 inclusion. (A) Diagram showing the location of the RRE stem-loop sequence. (B) RT-PCR analysis using RNAs isolated from transfected CA77 cells that preferentially exclude exon 4. CA77 cells were transfected with a RRE-containing reporter (lanes 1–5) in the absence of any protein plasmids (lanes 1) or the presence of 1 and 2 µg of Rev (lanes 2 and 3) or TIA-1-Rev (lanes 4 and 5). (C) RT-PCR analysis using RNAs isolated from CA77 cells transfected with the RRE-containing reporter in the absence of any proteins (lane 1) or the presence of 0.4 µg of Rev (lane 2) or TIA-1-Rev (lanes 3–9) together with 0.2–0.4 µg of HuC (lanes 4 and 5), Rev (lanes 6 and 7) or HuC-Rev (lanes 8 and 9). The bottom panel in B and C shows a bar graph indicating the level of the calcitonin exon inclusion upon overexpression of the transfected proteins.

When we cotransfected a construct containing the mouse HuC RRM3 together with the hinge domain with the calcitonin/CGRP reporter gene into CA77 cells, the production of CGRP was compromised and inclusion of the calcitonin exon was increased by approximately twofold compared with cells transfected without any Hu proteins (Figure 6, A and B, compare lane 1 with lanes 4 and 5. Transfection of the CA77 cells with the mHuC RRM12 construct did not significantly affect inclusion of the calcitonin exon (Figure 6, A and B, compare lane 1 with lanes 2 and 3).

View larger version (39K): [in this window] [in a new window]   Figure 6. Neuronal exclusion of exon 4 in CA77 cells is compromised by a truncated mHuC protein containing RRM3 domain with the hinge domain. (A) Diagram of the two truncated forms of mHuC is shown in the top panel. (B) CA77 cells were cotransfected with a calcitonin/CGRP reporter and increasing amounts (2 and 4 µg) of either mHuC RRM12 or mHuC RRM3 plasmid. The top panel shows an RT-PCR assay of total RNA from transfections of CA77 cells with the wild-type calcitonin/CGRP reporter gene and no mHuC (lane 1), increasing amount of mHuC RRM12 (lanes 2 and 3), or mHuC RRM3 (lanes 4 and 5). The middle panel shows Western blot analysis by using anti-Xpress antibody to indicate the level of the overexpressed mHuC proteins in HeLa cells. The bottom panel shows a bar graph indicating the level of the calcitonin exon inclusion upon overexpression of the transfected proteins.

Hu Proteins Interact with Endogenously Expressed Calcitonin/CGRP pre-mRNA
We took advantage of the fact that Hu proteins and calcitonin/CGRP are both endogenously expressed in CA77 cells and tested whether they interact in the natural environment of these cells. We immunoprecipitated the Hu protein-containing complex from a CA77 cell nuclear extract by using anti-Hu sera and examined the RNA in the complex by RT-PCR analysis. As shown in Figure 7, significant association of calcitonin/CGRP pre-mRNA was present in the immunoprecipitate, whereas no GAPDH pre-mRNA was brought down. PhosphorImager measurement of radioactivity in the RT-PCR products indicates that 55% of the imput calcitonin/CGRP pre-mRNA was pulled down by the anti- Hu sera. The strong association of Hu proteins and calcitonin/CGRP pre-mRNA supports the role of Hu proteins in regulating the neuron-specific RNA processing of this pre-mRNA.

View larger version (23K): [in this window] [in a new window]   Figure 7. Association of Hu proteins and the calcitonin/CGRP pre-mRNA in CA77 cells. The nuclear extract prepared from CA77 cells was immunoprecipitated with anti-Hu sera. RNA was isolated from either total nuclear extract or immunoprecipitated pellet and analyzed by RT-PCR. Minus RT lanes are included as controls. The oligonucleotides used to amplify the calcitonin/CGRP pre-mRNA anneal to sequences in intron 4, and the resulting PCR product is 225 nnucleotides, whereas the oligonucleotides used to amplify the GAPDH pre-mRNA anneal to sequences in intron 3 and exon 5, and the resulting PCR product is 585 nucleotides.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It seems that the major function of Hu proteins upon binding to the intronic U-tract sequence in neuronal or neuron-like cells, such as CA77 cells, is to block the activity of TIA-1/TIAR proteins, which would otherwise promote the inclusion of the calcitonin exon 4 (Figure 5C). Of particular note is the fact that in the experiment where TIA-1-Rev fusion protein is cotransfected with the RRE-containing reporter construct into CA77 cells, Hu proteins are presumably still able to bind the U-tract immediately downstream of the RRE stem-loop structure. However, such binding does not impact the inclusion of the calcitonin exon if TIA-1-Rev is present. Likewise, increasing amounts of HuC protein did not change the outcome either if TIA-1-Rev is present (Figure 5C).

If the major function of Hu proteins is to block TIA-1 and TIAR proteins, why does the RRM3 domain, but not the RRM1 and RRM2 domains together, have a dominant-negative effect? The lack of a dominant-negative effect by RRM1 and RRM2 can be explained by the fact that this protein, although binding to RNA specifically, has lowered binding affinity as judged by RNA gel-shift analysis (our unpublished data). Consistent with our results, the study by Chung et al. (1996) demonstrated that the RNA-binding affinity of the HuD full-length protein is almost 8 times greater than that of the HuDRRM1+ 2 protein. It is unclear at present how the RRM3 and hinge domains of Hu proteins functions as a dominant-negative protein. However, a hint came from the following studies. In one study, RRM3 of the human HuB protein interferes with the multimerization of the full-length proteins (Gao and Keene, 1996). Two other studies indicated that HuD as well as ELAV, the Drosophila homologue of Hu proteins, exist as multimers (Kasashima et al., 2002; Soller and White, 2005). The study on ELAV further demonstrated that binding of the multimerized ELAV on ewg RNA is functionally important (Soller and White, 2005). Thus, it is very tempting to suggest that multimerzation of Hu proteins is important for their functions as RNA processing factors, and overexpression of the RRM3 and hinge domains interferes with multimerization of the full-length proteins, which causes its dominant-negative effect.

Although in RNA gel mobility shift assay, recombinant proteins of the three neuron-specific Hu proteins show strong affinity for the calcitonin/CGRP intronic element containing the U-rich sequence (our unpublished data), our cell transfection experiments did not distinguish effect by individual Hu proteins (Figures 5 and 6). In CGRP-producing neurons, including hippocampus, dorsal root ganglia, and spinal cord, all three of the neuron-specific Hu proteins, HuB, HuC, and HuD, are present in adult mice (Okano and Darnell, 1997). A recent study using HuD-deficient mice demonstrated that HuD is involved at multiple stages during neuronal development. However, no difference of expression of several Hu protein targets was detected, suggesting at least a partial functional redundancy of Hu family proteins (Akamatsu et al., 2005).

The precise role of TIA-1/TIAR in promoting the nonneuronal inclusion of calcitonin/CGRP exon 4 is not clear. We propose that TIA-1/TIAR may play one of the two roles: inhibit recognition of the 3' splice site of exon 5, or enhance recognition of the 3' splice site of exon 4 (Zhu et al., 2003). In addition to the function mediated by intronic element, TIA-1/TIAR have been shown to promote authentic 5' splice site recognition by U1 snRNP (Del Gatto-Konczak et al., 2000; Forch et al., 2000; Le Guiner et al., 2001). In those examples, the TIA-1/TIAR binding sites are located immediately downstream of suboptimal 5' splice sites. Recent studies demonstrate an emerging theme of regulated alternative splicing by competing activities of TIA-1 and PTB. In one example, the two proteins bind to two different RNA sequences on the Fas pre-mRNA and have opposing activities in regulating the fate of Fas exon 6 (Izquierdo et al., 2005). In the other two examples, TIA-1 and PTB compete for binding at the same intronic sequences following the 5' splice sites (Zuccato et al., 2004; Shukla et al., 2005). The competing nature of binding of Hu and TIA-1/TIAR proteins at similar target sequence may be a wide spread phenomenon of neuron-specific alternatively spliced exons. Conceptually, it is likely that a subset of alternatively spliced exons is controlled by the competing activity of these two groups of proteins. However, at this point, it remains possible that other AU-rich sequence-binding proteins that we did not examine may also be involved in regulated RNA processing.

Experiments reported in this communication reveal a functional role of Hu proteins in regulating the neuron-specific alternative RNA processing of the calcitonin/CGRP pre-mRNA. However, although Hu proteins are necessary for the regulation, they are not sufficient. In HeLa cells, overexpression of Hu proteins, alone or in combination, did not switch the RNA processing phenotype (our unpublished data). This result suggests that other proteins may also be required for regulation in neurons. There is precedent that more than one neuron-specific protein is needed to include an alternative exon. At least two neuron-enriched proteins, nPTB and Fox-1, are required for inclusion of the c-src N1 exon in neurons (Markovtsov et al., 2000; Underwood et al., 2005).

Experiments using cell lines, embryos, and knockout mice demonstrated that Hu proteins are involved in early neuronal differentiation (Wakamatsu and Weston, 1997; Akamatsu et al., 1999, 2005; Anderson et al., 2000). Because some of the neuron-specific members of the Hu protein family have been shown to regulate mRNA stability and translation, it was postulated that the function of Hu proteins during neuron differentiation is to stabilize or promote translation of those mRNAs involved in neuronal function, such as the growth-associated protein-43 and neurofilament M, through binding at the AU-rich element located in 3' UTR (Antic et al., 1999; Anderson et al., 2000). Our results define the first nuclear function of Hu proteins and therefore suggest a novel mechanism by which Hu proteins regulate neuronal differentiation. It is entirely possible that Hu proteins regulate expression of genes associated with neuronal differentiation or maintenance by affecting alternative splicing of their pre-mRNAs. It is therefore of great importance to identify additional targets of Hu proteins and study the role of Hu proteins in regulating alternative splicing of these Hu target-containing pre-mRNAs. Our initial search of the alternative splicing database identified several neuron-specific alternatively spliced exons that are surrounded by U-rich sequences. Experiments are under way to investigate the role of Hu proteins in regulating these alternative splicing events.

Our studies reported here add Hu proteins to the currently very short list of tissue-specific RNA processing regulators. Additional studies of these proteins will provide significant insight into the regulatory mechanisms of neuron-specific alternative RNA processing.

	ACKNOWLEDGMENTS

 
We thank the following individuals for providing cell lines, antibody, and plasmids: Alison Hall and Andrew Russo (CA77), Lloyd Culp (SK-N-SH), Gary Landreth (PC12), Jerome Posner (Hu patient sera), Ite Laird-Offringa (HuD plasmid), and Christine Herrmann (PC12-Rev). We thank Helen Salz, and Jo Ann Wise for critical reading of the manuscript. This work was supported by National Science Foundation Grant MCB-0237685 and National Institutes of Health Grant to H.L. (NS-049103-01). H.Z. is supported by a predoctoral fellowship from American Heart Association.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-02-0099) on October 11, 2006.

Address correspondence to: Hua Lou (hxl47{at}case.edu)

Abbreviations used: CGRP, calcitonin gene-related peptide.

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