Binding of p190RhoGEF to a destabilizing element on the light neurofilament mRNA is competed by BC1 RNA.

The enhancement of RNA-mediated motor neuron degeneration in transgenic mice by mutating a major mRNA instability determinant in a light neurofilament (NF-L) transgene implicates cognate RNA binding factors in the pathogenesis of motor neuron degeneration. p190RhoGEF is a neuron-enriched guanine exchange factor (GEF) that binds to the NF-L-destabilizing element, to c-Jun N-terminal kinase-interactive protein-1 (JIP-1), and to 14-3-3 and may link neurofilament expression to pathways affecting neuronal homeostasis. This study was undertaken to identify additional RNA species that bind p190RhoGEF and could affect interactions of the exchange factor with NF-L transcripts. The C-terminal domain of p190RhoGEF, containing the RNA-binding site, was expressed as a glutathione S-transferase fusion protein and was used as an affinity probe to isolate interactive RNAs in rat brain extracts. As expected, NF-L mRNA was identified as an RNA specie eluted from the affinity column. In addition, BC1 RNA was also found enriched in the bound RNA fraction. BC1 is a 152-nucleotide RNA that is highly expressed but untranslated in differentiated neurons. We show that BC1 and NF-L mRNA bind to a similar site in the C-terminal domain of p190RhoGEF, and their bindings to p190RhoGEF are readily cross-competed. Moreover, we identify a novel binding site in BC1 to account for its interaction with p190RhoGEF. The findings suggest a novel role of BC1 in differentiated neurons involving RNA-protein interactions of p190RhoGEF.

Motor neuron degeneration occurs in transgenic mice expressing high levels of a neurofilament light subunit (NF-L) 1 transgene (1) or neurofilament heavy subunit transgene (2) but not in mice expressing high levels of a neurofilament mid-sized subunit transgene (3). A more severe form of motor neuron degeneration occurs in mice expressing low levels of an NF-L transgene with a leucine-to-proline mutation in the rod domain and a c-Myc tag appended to the C-terminal end of the protein (4). A severe form of motor neuron degeneration also occurs from low level expression of an NF-L transgene lacking the point mutation but with the c-Myc tag at the end of the coding region (5). The latter finding raised the possibility that neuropathic effects result from expression of a mutant mRNA by the transgene. This interpretation is based on the location of the c-Myc tag in a major destabilizing element in NF-L mRNA and the ability of the c-Myc insert to stabilize the transcript as well as alter the binding of cognate binding factors in brain extracts to the NF-L mRNA destabilizing element by gel-shift and crosslinking assays (5). More recently, motor neuron degeneration was reproduced in transgenic mice when the destabilizing element of NF-L mRNA was placed in the 3Ј-untranslated region of an enhanced green fluorescent protein reporter transgene (6).
p190RhoGEF was identified as a potential component in RNA-mediated motor neuron degeneration by virtue of its ability to bind to the stability determinant and alter NF-L mRNA stability (7) as well as its interaction with JIP-1 (8), microtubules (9), and 14-3-3 (10). More recently, we have shown that overexpression of p190RhoGEF protects Neuro-2a cells from stress-induced apoptosis, possibly by interacting in the JIP-1/ c-Jun N-terminal kinase pathway. 2 Moreover, p190RhoGEF is a neuronal enriched exchange factor that is up-regulated during postnatal development and highly expressed in large differentiated neurons, including motor neurons in mouse spinal cord. 2 A similar expression profile is exhibited by NFs (11) and by neuron-enriched isoforms of JIP-1 (12) and 14-3-3 (13)(14)(15).
The involvement of the NF-L-destabilizing element in RNAmediated motor neuron degeneration raised the possibility that additional RNA-protein interactions of the C-terminal domain of p190RhoGEF may be instrumental in modulating RNAmediated neuropathic effects. To pursue this line of study, the C-terminal domain of p190RhoGEF (p190RhoGEF-C) containing the RNA-binding site was fused to GST protein and used as affinity matrix for identifying additional interactive RNA species in soluble brain extracts. To our surprise, we identified BC1 as an RNA with high affinity for the RNA-binding site in p190RhoGEF. BC1 is an untranslated 152-nucleotide polymerase III transcript that is up-regulated during postnatal development (16) and highly expressed in large neurons of rat brain and spinal cord (17,18). The importance of BC1 in neuronal function is not yet fully defined, but the favored view is that BC1 serves as a molecular scaffold for regulating transport and translation of neuronal mRNAs at dendritic sites (19,20). The binding of BC1 and NF-L mRNA to the same binding site in p190RhoGEF, as reported herein, suggests that BC1 may play a role in RNA-protein interactions of p190RhoGEF and in modulating the effects of the exchange factor in large differentiated neurons. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
DNA Constructs-A cDNA-encoding sequence between amino acids (aa) 1276 -1582 in the C-terminal domain of p190RhoGEF was fused in-frame to glutathione S-transferase (GST) in pGEX-6P (Amersham Biosciences) and is referred to as GST/p190RhoGEF-C. The same cDNA was cloned into pHM6 (Roche Molecular Biochemicals) for HA tagging and was modified by PCR to generate N-and C-terminal truncations. All cDNA constructs were verified by DNA sequencing.
Isolation of RNA from Brain Extracts Using GST Fusion Proteins-GST/p190RhoGEF-C and unfused GST proteins (100 g) were bound to 40-l glutathione-Sepharose beads and equilibrated with RNA-binding buffer (RBB) containing 10 mM Hepes, pH 7.5, 50 mM KCl, 3 mM MgCl 2 , 2 mM dithiothreitol, and 5% glycerol. High speed supernatant (200 l) of rat brain homogenate was precleared by passage through an unfused GST column and then applied to a column of immobilized GST fusion proteins. The column was washed with RNA-binding buffer containing heparin (1 mg/ml), and RNA was recovered by boiling the beads in 0.1% SDS. Supernatants were phenol/chloroform-extracted, and RNAs were ethanol-precipitated.
cDNA Library Construction from Extracted RNA-cDNA libraries were constructed from total RNA (50 ng) using the SMART cDNA library construction kit (Clontech). Individual phage plaques were amplified, DNAs extracted, and cDNAs sequenced.
Preparation of RNA Probes-RNA polymerase templates for fulllength and truncated BC1 probes were prepared as PCR products by incorporating T7 promoter sequence into the upstream primer. A probe to the 68-nucleotide destabilizing element in NF-L (NF68) was obtained from a linearized pSKϩ plasmid (7). Probes were uniformly labeled with [␣-32 P]UTP and fractionated by electrophoresis, and gel-excised probes were diluted in RBB to 5 ϫ 10 4 cpm/l immediately prior to use.
Gel-shift Studies-Gel-shift studies were conducted by incubating 10 g of protein and 5 ϫ 10 4 cpm/ml radioactive probe in 20 l of RBB at room temperature for 10 min, then adding 2 l of RNase T1 (2 units/l), and incubating for an additional 10 min. Five l of heparin (50 mg/ml in 50% glycerol) was added, and the total sample was loaded onto a 10% acrylamide gel and electrophoresed at 100 V in 0.5ϫ TBE. Cell Culture and Transfection-Neuro-2a cells were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cell transfections were performed using the FuGENE6 reagent (Roche Molecular Biochemicals).
Immunoprecipitation Assays-Transfected N2a cells in 100-mm cell culture plates were detached and collected by centrifugation, washed with PBS, and suspended in 0.6 ml of cell lysis buffer (1ϫ PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 0.01% (v/v) of Protease Inhibitor Mixture (Sigma), dispersed by centrifugation in a QIA shredder Mini column (Qiagen). Extracts were mixed with 20 l of anti-HA antibody-agarose conjugate (Santa Cruz Biotechnology) on a shaker for 1 h at 4°C, and beads were precipitated by centrifugation, washed 4 times with PBS, suspended in 20 l of 0.1% SDS, and boiled for 3 min to release bound components. The supernatant was then used as a template for RT-PCR detection of BC1 RNA.
Northwestern Blot-GST fusion proteins (10 g) were electrophoresed in 8% SDS-PAGE gel and electroblotted to Hybond-C membrane (Amersham Life Science). Membranes were preincubated with Blotto A (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% milk) on shaker at 4°C overnight and incubated with 5 ϫ 10 4 cpm/ml probe in hybridization buffer for 1 h at room temperature. Membranes were extensively washed with hybridization buffer, and dried membranes were subjected to overnight exposure for autoradiography.

BC1 and NF68 Bind to and Cross-compete for
p190RhoGEF-The C terminus of p190RhoGEF (p190 Rho-GEF-C) was isolated from a rat brain cDNA library by virtue of its binding to a 68-nucleotide RNA probe (NF68) comprising a destabilizing element in mouse NF-L mRNA (7). To identify additional RNA targets of p190RhoGEF-C, the cDNA was fused to GST, and fusion protein was used as an affinity matrix to screen a high speed supernatant fraction of rat brain. Bound RNAs were recovered from the beads, reverse-transcribed into cDNAs, and the cDNAs cloned and sequenced. The resulting cDNAs not only included NF-L but also revealed more frequent recovery of short elements matching the sequence of BC1. The structure and partial sequence of BC1 is shown in Fig. 1.
To verify the binding of BC1 to p190RhoGEF-C, GST/ p190RhoGEF-C (150 pmol) was spotted on nylon membrane, along with equimolar amounts of unfused GST protein (GST) and bovine serum albumin (BSA), and reacted with uniformly labeled full-length BC1 probe (Fig. 2). BC1 bound to p190RhoGEF-C but not to BSA or to unfused GST protein. A similar-sized RNA probe to sequence adjoining the T7-binding site in pSK ϩ (SKϩ) did not bind to GST/p190RhoGEF-C, GST, or BSA. Moreover, binding of BC1 was more than 10-fold greater than binding of NF68 to p190RhoGEF-C by comparing the respective amounts of bound radioactivities. Similar disparity in binding affinities were observed using probe to the ID sequence of BC1 (data not shown), suggesting that the binding site on BC1 was situated within the ID region of BC1 RNA (see below).
Gel retardation assays were undertaken to assess the abilities of BC1 and NF68 probes to alter the electrophoretic migration of GST/p190RhoGEF. Fig. 3 shows that incubation of GST/p190RhoGEF-C with 32 P-labeled NF68 or BC1 probes generates similar radioactive bands (arrow). The radioactive gel-retarded band formed with NF68 (3rd lane) was competed by 100-fold excesses of unlabeled NF68 (4th lane) or unlabeled BC1 (5th lane) but not by an unlabeled nonspecific SKϩ probe (data not shown). Likewise, the gel-retarded band formed with BC1 (6th lane) was competed by 100-fold excesses of unlabeled NF68 (7th lane) or unlabeled BC1 (8th lane) but not by an unlabeled SKϩ probe (data not shown). Incubation of GST/ p190RhoGEF with 32 P-labeled SKϩ probe did not generate a gel-retarded band (9th lane).
In Vivo Binding of BC1 RNA to p190RhoGEF Protein-Lysates from Neuro-2a cells transfected with HA-tagged p190RhoGEF-C protein or pHM6 vector alone were immuno- p190RhoGEF Binding to BC1 and NF-L RNA precipitated with agarose-conjugated anti-HA monoclonal antibody. The immunoprecipitates were washed extensively and solubilized by boiling in 0.1% SDS. RT-PCR assays were conducted on the solubilized fractions to assess the presence of BC1 RNA in the respective immunoprecipitates. Fig. 4 shows that BC1 RNA (arrow) was recovered from Neuro-2a cells transfected with HA-tagged p190RhoGEF-C (lane 1) but not from Neuro-2a cells transfected with the empty pHM6 vector (lane 2). The size of the BC1 RT-PCR fragment was 175 bp, including T7 promoter sequence in upstream primer, and had the appropriate electrophoretic migration when compared with the 100-bp electrophoretic standard markers (lane M).
Binding Site on p190RhoGEF Protein for BC1 RNA-Fulllength and truncated GST/p190RhoGEF-C were constructed for localizing the NF68-binding site by Northwestern blots. 3 The same full-length and truncated GST fusion proteins were used to localize and compare the BC1-and NF68-binding site (Fig. 5A). GST fusion proteins were expressed in bacteria and purified by glutathione affinity chromatography. Purified proteins were electrophoresed in SDS-PAGE (Fig. 5B) and transferred to nylon membranes, and replicates of transferred proteins were hybridized with BC1 (Fig. 5C) and NF68 (Fig. 5D) probes by Northwestern blots. Full-length and N-terminal truncations (M4 and M5 constructs) of GST/p190RhoGEF-C bound 32 P-labeled BC1 (Fig. 5C) and NF68 (Fig. 5D) probes. Binding of both BC1 and NF68 probes was lost in C-terminal truncations of GST/p190RhoGEF-C. Whereas binding was retained when the C terminus of GST/p190rhoGEF-C was truncated from aa 1582 to 1524 (M3 construct), binding of NF68 and BC1 probes was eliminated upon C-terminal truncations to aa 1493 (M2 construct) and 1461 (M1 construct). The findings indicate that BC1 and NF68 bind to similar sites in GST/ p190RhoGEF and that sequence encoded between aa 1493 and 1524 (QLQEYQQSLERLREGQRMVERERQKMRVQQGL) is required for the binding of both RNA probes.
Binding Site on BC1 RNA for GST/p190RhoGEF-C-GST/ p190RhoGEF-C was separated by SDS-PAGE and transferred to nylon membrane for Northwestern blots to determine the sequence in BC1 required for binding to the fusion protein.
Membrane containing fusion protein was hybridized with fulllength BC1 and with a series of truncated probes lacking 5Ј or 3Ј sequences (Fig. 6A). 32 P-Labeled probes to full-length BC1 (BC1), to ID sequence (ID), and to the full-length BC1 probe lacking the initial 10 nucleotides of the dendrite-targeting motif or DTM (BC1⌬DTM) bound to the fusion protein (Fig. 6B). Binding of 32 P-labeled probes to the fusion protein was lost when the 5Ј deletion was extended to include the initial 25 (BC1⌬ID-A) or 50 (BC1⌬ID-A/B) nucleotides of BC1. The findings indicated that sequence within the ID element of BC1 was sufficient for binding and that sequences in the initial 25 nucleotides of BC1, excluding the DTM, are necessary for binding of BC1 to GST/p190RhoGEF-C. p190RhoGEF Binding to BC1 and NF-L RNA DISCUSSION Novel binding features of BC1 are herein identified which expand the prospective role of BC1 in neuronal metabolism. Binding of BC1 and the destabilizing element in NF-L mRNA to a similar site in the C-terminal domain of p190RhoGEF links BC1 to NF-L expression and to RNA-mediated motor neuron degeneration (7). The recognition of p190RhoGEF as an RNAbinding protein also links NF-L expression with signal transduction pathways and with potential modifications of the neuronal cytoskeleton in differentiated neurons (21)(22)(23). Moreover, the identification of a novel binding site outside of the dendritetargeting motif (DTM) supports the likelihood that BC1 has additional functional properties beyond its putative role in regulating movement, localization, and translation of neuronal transcripts in the dendritic compartment (19,20).
There is presently much more information as to the derivation than function of BC1. For example, BC1 is recognized as a master gene for generating and amplifying short interspersed repetitive elements in the rodent genome (24 -26) analogous to the Alu repeats in the primate genome (27)(28)(29). BC1 and other progenitor genes are believed to have arisen by reverse transcription and by fortuitous integration of cDNAs into a favorable site of the genome downstream of a pol III promoter sequence. BC1 is, however, an atypical pol III transcript in that it is highly and exclusively expressed in differentiated neurons (30). Neuron-specific transcription is regulated by positive and negative upstream elements that react with brain-specific and nonspecific factors (18). Competitive binding of Pur protein to the BC1 transcript and to cis-acting enhancer elements in the BC1 gene provides a potential feedback mechanism for regulating BC1 transcription by its own gene product (31).
The up-regulation of BC1 during the late phase of neuronal differentiation and high level expression in large differentiated neurons (16) coincides with the expression profiles of NF-L (11) and p190RhoGEF 2 and is consistent with a functional interaction of these components in large differentiated neurons. A similar expression profile is also characteristic of interactive partners of p190RhoGEF, including neuron-enriched isoforms of JIP-1 (12) and 14-3-3 (13)(14)(15). It is possible that binding of one or more of the interactive partners to the C-terminal domain could be responsible for unmasking inherent GDP/GTP activity required for activation of p190RhoGEF in vivo (9). It is also possible for interaction of BC1 with p190RhoGEF to account for axonal transport or localization of BC1 in discrete sites along the surface membrane of goldfish Mauthner axons (32). It should be noted, however, there are still large regions in the N-terminal half of p190RhoGEF containing potential inter- p190RhoGEF Binding to BC1 and NF-L RNA active sites of unknown function, including zinc finger and leucine-rich motifs (33).
The putative function of BC1 in regulating dendritic expression is based on the properties of specific BC1-bound proteins. Likewise, our fortuitous identification of additional BC1-binding proteins suggests that BC1 may have additional interactive roles in neuronal gene expression. In both instances, it is proposed that BC1 functions as a RNA-protein complex and that RNA binding confers, alters, or modifies properties of the complex. Such RNA-protein interactions resemble the interactions of multiple RNAs and proteins in RNP complexes that mediate the processing of RNA (34,35). More recently, it is also recognized that short single-stranded RNAs can regulate gene expression by hybridizing with specific mRNAs as small interfering or small temporal RNAs (36 -38).
The potential involvement of p190RhoGEF in RNAmediated motor neuron degeneration is quite unexpected because the function of p190RhoGEF has been attributed to regulation of the neuronal cytoskeleton (8). It should be noted, however, that another GEF, i.e. RCC1, plays a critical role in regulating the translocation of protein and RNA across the nuclear pore complex by promoting the formation of Ran-GTP on the nuclear face of the complex (39). More recently, a GEF protein with close homology to RCC1 was identified as the mutant gene product in a recessive form of familial motor neuron degeneration (40,41). Why dysfunction of a GEF should lead to motor neuron degeneration is also unexpected. The role of RNA-protein interactions on p190RhoGEF and their potential linkage to RNA-mediated motor neuron degeneration could provide some insights into the nature of motor neuron disease. p190RhoGEF Binding to BC1 and NF-L RNA