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Published as doi: 10.1096/fj.07-9532com.
 (The FASEB Journal. 2008;22:691-702.)
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(The FASEB Journal. 2008;22:691-702.)
© 2008 FASEB

A novel noncoding RNA rescues mutant SOD1-mediated cell death

Yueming Chang*,{dagger}, Michael P. Stockinger*, Hirofumi Tashiro* and Chien-liang Glenn Lin*,{dagger},1

* Department of Neuroscience,

{dagger} Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, USA

1Correspondence: The Ohio State University, Department of Neuroscience, 4198 Graves Hall, 333 W. 10th Ave., Columbus, Ohio 43210, USA. E-mail: lin.492{at}osu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing mutant Cu2+/Zn2+ superoxide dismutase SOD1(G93A) develop similar clinical and pathological phenotypes to amyotrophic lateral sclerosis (ALS) patients. Here, we utilize representational difference analysis to identify the transcripts that are up-regulated in the presymptomatic stage of SOD1(G93A) mice. Unexpectedly, three predominant clones were 18S or 28S ribosomal RNA (rRNA) segments. One of these clones corresponded to a capped and polyadenylated transcript containing a large portion of 18S rRNA, named MSUR1 (mutant SOD1-up-regulated RNA 1). In vitro expression experiments show that MSUR1 is able to rescue SOD1(G93A)-mediated cell death. Expression of MSUR1 significantly reduces SOD1(G93A)-induced free radical levels and oxidative damage. Further, MSUR1 can reduce hydrogen peroxide-mediated cytotoxicity. MSUR1 does not encode a protein, suggesting its role as a functional noncoding RNA. It is widely expressed in various tissues. Searching the database of GenBank revealed that a large number of expressed sequence tag (EST) clones contain large portions of rRNA sequence, potentially indicating a heretofore overlooked class of mRNAs with functional significance.—Chang, Y., Stockinger, M. P., Tashiro, H., Glenn Lin, C. A novel noncoding RNA rescues mutant SOD1-mediated cell death.


Key Words: ALS • neurodegeneration • rRNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AMYOTROPHIC LATERAL SCLEROSIS (ALS) is a fatal neurodegenerative disorder that is clinically characterized by muscle wasting, weakness, and spasticity, reflecting the relatively selective degeneration of motor neurons in the spinal cord, motor cortex, and brainstem, which typically results in mortality within 2–5 yr after the onset of disease (1) . The majority of cases have no genetic component. Approximately 5% of ALS cases are familial (FALS) with an autosomal dominant inheritance pattern. About 15–25% of the cases of FALS are linked to mutation in the gene encoding the antioxidant enzyme Cu2+/Zn2+ superoxide dismutase (SOD1). The causes for most cases of ALS are unknown. It is believed that multiple factors underlie the disease mechanism (2 3 4) .

Overexpression of some FALS-linked mutant SOD1 proteins (i.e., G93A, G37R, and G85R) in transgenic mice results in the development of a neurological disorder that resembles ALS (5 6 7 8) . Mutant SOD1 causes motor neuron degeneration by acquiring a toxic gain of function property, rather than by the loss of enzymatic activity (9 10 11) . The molecular mechanisms underlying mutant SOD1-linked FALS remain unclear. Many studies have shown that the mutant SOD1 toxicity to motor neurons is noncell autonomous, meaning that mutant damage is required within both motor neurons and non-neuronal cells in order to fully represent the ALS phenotype (12 13 14 15 16) .

The transgenic mouse expressing mutant SOD1(G93A) is a commonly used ALS animal model (6) . These mice show obvious loss of motor neurons and associated motor function at ~3 months of age and eventually die at ~4–5 months of age. There is no motor neuron loss at 2 months of age. The original idea of this study was that there may be factors induced at an early age having protective functions against mutant SOD1(G93A) toxicity. We performed representational difference analysis (RDA) (17) , a polymerase chain reaction (PCR) -based subtraction hybridization method, and identified a novel transcript named MSUR1, which was up-regulated in early age (1–2 months) of SOD1(G93A) mice. The data presented here show that MSUR1 is a member of a group of polyadenylated transcripts that contain large portions of 18S or 28S ribosomal RNA (rRNA) sequence. Importantly, MSUR1 is capable of reducing mutant SOD1(G93A)-mediated toxicity and cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
SOD1(G93A) [strain B6SJL-TgN(SOD1-G93A)1Gur] and wild-type SOD1 [strain B6SJL-TgN(SOD1)2Gur] as well as nontransgenic (strain B6SJLF1/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

RDA
RDA was conducted with cDNAs derived from poly(A)+ RNA prepared from the spinal cord of SOD1(G93A) transgenic or nontransgenic sibling mice according to Hubank and Schatz (17) except that the third and final hybridization was carried out at a tester:driver ratio of 1:100,000 instead of 1:400,000. The transgenic SOD1(G93A) mouse sample was used as the tester, and the nontransgenic mouse sample was used as the driver.

Poly(A)+ RNA isolation
Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol. Poly(A)+ RNA was selected from total RNA using the Oligotex mRNA Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s directions. To ensure that all possible nonpoly(A)+ RNA were removed, RNA was passed through the selection process twice and washing steps were increased in number from two to three.

Reverse transcription (RT) and 3' rapid amplification of cDNA ends (3' RACE)
1st strand cDNA was synthesized from poly(A)+ RNA reverse transcribed using the Thermoscript RT-PCR system (Invitrogen) according to manufacturer’s protocol. PCR was performed at a final concentration of 1x PCR buffer/3 mM MgCl2/0.3 mM dNTPs/0.4 µM each 5' and 3' primers/10% DMSO/2 µl of RT reaction mixture in a total of 50 µl. The mixture was amplified with an MJ Research PTC-200 thermal cycler for 30 cycles. Initial denaturation was at 98°C for 5 min, followed by 20 s at 75°C during which 2.5 U Taq polymerase (Invitrogen) were added to each sample. Initial annealing was 2 min at 55°C followed by 40 min at 72°C. This was followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and primer extension at 72°C for 3 min. Final extension was at 72°C for 15 min. Primers used for PCR were as follows: primer B (5'-GGTCTGTGATGCCCTTAGATGTCCG-3') corresponding to position +1490 of 18S rRNA gene; primer E (5'-GATGTCGGCTCTTCCTATCATTGTGAA-3') corresponding to position +4098 of 28S rRNA gene; primer T7-(dT)24 (5'-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCTTTTTTTTTTTTTTTTTTTTTTTT-3'); and primer Q1 (5'-GTGAATTGTAATACGACTCACTATAGGCGC-3').

To examine nonspecific binding of T7-(dT)24 to stretches of adenine, RT was performed as described above using total RNA. Primers used were T7-(dT)24, Q1, primer A (5'-GGGTGACGGGGAATCAGGGTTC-3') corresponding to position +404 of 18S rRNA gene, and primer C (5'-CAGTCGGTCCTGAGAGATGGGC-3') corresponding to position +2191 of 28S rRNA gene. PCR conditions were the same as described above.

To examine whether the transcripts corresponding to the identified PCR products (Fig. 1 B) contain the RDA fragment regions, the same RT product prepared from poly(A)+ RNA was used. Primers used were primer A, primer +313 (5'-GATCGCACGCCCCCCGTG-3') corresponding to position +313 of 18S rRNA gene, primer F (5'-GAGCAGTTTTAATGAGGGTGCAGTGTGTAC-3') corresponding antisense to the junction between 18S rRNA gene sequence and the 3' unknown sequence region, and primer G (5'-GGCATTGAGACCAGGCACAGTATC-3') corresponding antisense to the 28S unknown sequence region. PCR conditions were the same as described above.


Figure 1
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  		Figure 1. Three clones identified by RDA correspond to capped and polyadenylated RNAs that contain large portions of rRNA sequence. A) Positions of the 3 identified clones corresponding to rRNA and positions of primers used for reverse transcription (oligo dT-T7) and PCR (A–E and Q1). (A)6 and (A)4 indicate positions of stretches of adenine within the rRNA. B) RT-PCR analyses of mRNA prepared from SOD1(G93A) or nontransgenic spinal cords using primers B/Q1 and E/Q1. C) Sequence structures of transcripts corresponding to 18S and 28S-2 clones (GenBank accession nos. AY248756 and AY248755). Double underlines indicate polyadenylation signals. USR, unknown sequence regions

5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE)
The 5' RLM-RACE was performed using the First Choice RLM-RACE Kit (Ambion, Austin, TX, USA) according to manufacturer’s protocol. Briefly, poly(A)+ RNA was isolated from mouse cerebral cortex tissue as described above, processed with the kit enzymes to ligate the 5' RACE adapter to capped, full-length mRNA transcripts. 2 step nested PCR was then performed as described above protocol using kit provided as well as primers +530AS (5'-TTGTTATTTTTCGTCACTACCTCCCCG-3') corresponding antisense to position +530 of 18S rRNA gene for step 1 and primer +475AS (5'-CTGCCTTCCTTGGATGTGGTAGCC-3') corresponding antisense to position +475 of 18S rRNA gene for step 2.

Northern blot analysis
Five-hundred nanograms of poly(A)+ selected RNA were fractionated by electrophoresis through a 1.9% formaldehyde/1.2% agarose gel and transferred to positively charged nylon membranes (Roche, Indianapolis, IN, USA) by capillary action overnight in 10x standard saline citrate (SSC). RNA was fixed to the membrane by ultraviolet crosslinking twice and baking at 80°C for 30 min. Antisense RNA probes were labeled with digoxigenin (DIG)-11-UTP (Roche) by in vitro transcription using T7 RNA polymerase (USB, Cleveland, OH, USA) for 90 min. at 37°C according to the manufacturer’s protocol. Membrane was prehybridized 4 h at 65°C and hybridized 16 h at 65°C. Stringency washing conditions were 2' 5 min at 65°C in 1x SSC, 0.5% sodium dodecyl sulfate (SDS), 0.1% Sarkosyl followed by 2' 15 min at 65°C in 0.5x SSC, 0.1% SDS. Additional very high stringency washing conditions were 2' 15 min at 70°C in 0.2x SSC, 0.2% SDS. Antibody incubation and detection followed the manufacturer’s protocol (Roche). When duplicate blots were required, i.e., multiple tissue blots, individual mRNA samples were prepared in bulk and split to equal loading volumes to ensure equal loading of lanes. Equal loading was confirmed in each case by hybridization to a mouse β-actin probe transcribed from pTRI-Actin-Mouse (Ambion) corresponding to nucleotides 739–989 of GenBank accession no. X03672.

Transient transfection and Western blot analysis
HEK293 cells (American Type Culture Collection, Manassas, VA, USA) were transiently transfected with indicated plasmid DNA using LipofectAMINE PLUS (Invitrogen) as described previously (18) . Cells were harvested 72 h after transfection. NSC34 cells (kindly provided by N. Cashman, University of Toronto, Toronto, ON, Canada, and R. Liu, University of North Dakota, Grand Forks, ND, USA) were transiently transfected with indicated plasmid DNA using electroporation. In brief, after NSC34 cells were incubated with 10 µg plasmid DNA for 10 min at room temperature in 200 µl Opti-Mem (Invitrogen), they were electroporated under 250 V, 50 ms, 2 pulses by Electro Square Porator ECM 830 (BTX, San Diego, CA, USA). The transfected cells were then incubated at room temperature for 30 min and transferred to petri dishes. For Western blot analysis, protein extracts were generated from transfected cells, resolved by SDS-PAGE, and transferred onto PVDF membranes as described previously (19) . A rabbit anti-SOD1 pAb (1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. The immunoreactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer’s directions.

RT-PCR analysis
Total RNA were isolated as described above, and first-strand cDNA was synthesized with M-MLV reverse transcriptase (Invitrogen) using MSUR1-specific primer (5'-AGCAATTCAGAGCAGTTTTAATGAGGGTGC-3', corresponding to position +1701 of MSUR1 or 5'-GAGCAGTTTTAATGAGGGTGCAGTGTGTAC-3', corresponding to position +1692 of MSUR1). SOD1-specific primer (5'-CAGCAGTCACATTGCCCARGTCTCCAACATG-3', corresponding to position +349 of mouse SOD1) or β-actin-specific primer (5'-TGTCAAAGAAAGGGTGTAAAACGCAGC-3', corresponding to position +1223 of mouse β-actin) were used as controls. The following primers were used for PCR: MSUR1 F +1490 (5'-GGTCTGTGATGCCCTTAGATGTCCG-3') and R +1701 (5'-AGCAATTCAGAGCAGTTTTAATGAGGGTGC-3' or R +1692 5'-GAGCAGTTTTAATGAGGGTGCAGTGTGTAC-3'), SOD1 F +1 (5'-ATGGCGACGAAGGCCGTGTGCGT-3') and R +349 (5'-CAGCAGTCACATTGCCCARGTCTCCAACATG-3'), and β-actin F +626 (5'-TGTCAAAGAAAGGGTGTAAAACGCAGC-3') and R +1223 (5'-CGGGACCTGACAGACTACCTCAT-3'). PCRs were performed in the presence of 3 mM MgCl2, 0.2 mM dNTP, 0.25 µM primers, and 2 U TaqDNA polymerase (Invitrogen) in 1x PCR buffer. Twenty-five (for controls) or thirty-five (for MSUR1) cycles were performed (95°C for 30 s, specific annealing temperature to each set of primers for 45 s, and 72°C for 1 min). PCR products were visualized as single bands on 1% agarose gels stained with ethidium bromide.

Dichlorofluorescence (DCF) detection
The method for fluorescence detection of oxidative stress was slightly modified from the original as described by Wang and Joseph (20) . Cell cultures were grown at least 24 h before assay. Media were removed and replaced with 1x PBS containing 100 µM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Cultures were placed back into the incubator for 30 min followed by washing with PBS. Cultures were flooded with media containing 400 or 700 µM hydrogen peroxide at 37°C for 30 min and then placed in a multiwell fluorescence microplate reader maintained at 37°C. Culture fluorescence was followed for 30 min at an excitation 485 nm and an emission at 530 nm. Data points were taken every 5 min. After the final measurement at 30 min, the percentage of increase was determined using the formula [(FT30–FT0)/FT0x100], where FT0 = fluorescence at 0 min and FT30 = fluorescence at 30 min.

Protein oxidation assay
Protein extracts were incubated with 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2N HCl at room temperature for 1 h. One volume of 20% TCA were added to each sample and incubated for 30 min on ice to facilitate the protein precipitation. The precipitated proteins were collected by centrifugation at 4°C with maximum speed for 20 min, washed with cold acetone, air-dried, and then added 1x SDS loading buffer. Western blotting was performed and used anti-dinitrophenyl-keyhole limpet hemocyanin (KLH) antibodies to recognize DNPH binding oxidized protein (Invitrogen).

MTT assay
For the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cells were plated at least 48 h before experiment. Cells were rinsed with 1x PBS and then incubated with 25 µg/ml MTT in DMEM (Invitrogen) for 30 min at 37°C. The reaction was stopped by adding 1 ml of stop solution (0.04 M HCl in isopropanol). After the plate was shaken to completely dissolve crystals on the bottom of it, the absorbance of violet color was measured in a computer-controlled microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) at wavelengths of 570 nm.

Deletion constructs
For deletion 1, pcDNA3/MSUR1 was digested with BamHI and then religated to delete +1 to +591, resulting in missing ORF1–3. For deletion 2, pcDNA3/MSUR1 was digested with ApaI and then religated to delete +1 to +794, resulting in missing ORF1–3 and part of ORF4.

In vitro translation
MSUR1, deletion 1, SMN, and luciferase cDNAs in pcDNA3 vector were used as DNA templates for TNT Quick Coupled Transcription/Translation Systems in the presence of T7 RNA polymerase and [35S]methionine according to manufacturer protocols (Promega, Madison, WI, USA). The reactions were carried out at 30°C for 90 min. The synthesized protein products were electrophoretically resolved through a 7 M urea 18% SDS-PAGE Bis-Tris gel, which was able to resolve peptides of less than 2.5 kDa; the gel was then dried and exposed to autoradiography film for 24 h.

Site-directed mutagenesis
Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used according to the manufacturer’s protocol. The following single point mutations on the MSUR1 ORF4 and MSUR1 ORF5 were introduced via site-directed mutagenesis: T770A and T919A, respectively. Mutagenesis was performed with pcDNA3/MSUR1 as a template using the primers T770A sense primer (5'-CGCCCCCTCGATGCTCGATGCTGAGTGT-3'), T770A antisense primer (3'-GCGGGGGAGCTACGAGATTCGACTCACA-5'), T919A sense primer (5'-CGGAACTGAGGCCATGATTTAGAGGGACGG-3'), and T919A antisense primer (3'-GCCTTGACTCCGGTACTAAATCTCCCTGCC-5'). The mutations were confirmed by sequencing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The identified RDA clones correspond to the capped and polyadenylated transcripts containing large portions of rRNA sequence
RDA was performed using cDNAs derived from poly(A)+ RNA prepared from the spinal cords of SOD1(G93A) transgenic or nontransgenic sibling mice. The transgenic SOD1(G93A) mouse sample was used as the tester, and the nontransgenic littermate sample was used as the driver. The products resulting from RDA were expected to be up-regulated or newly induced mRNA in the spinal cord of SOD1(G93A) transgenic mice. We examined three different ages of mice including 1-, 2-, and 4-month-old mice. Among the identified clones, three caught our attention because they were the predominant clones found in the presymptomatic stage (1 and 2 months of age), and they corresponded to two related genes. Clone 1 corresponded to 18S rRNA (hereafter referred to as clone 18S), and clones 2 and 3 corresponded to two different parts of 28S rRNA (hereafter referred to as clone 28S-1 and clone 28S-2, respectively) as shown in Fig. 1A .

We first tested whether these clones were simply artifacts resulting from the oligo(dT) primer nonspecifically binding to the short stretches of adenine present in contaminating 18S or 28S rRNA during RT reaction. The results showed that these clones were not artifacts generated from RT of rRNA. We then examined the following two possibilities: 1) some portion of 18S or 28S rRNA may have poly(A) tails, and this subset is up-regulated in SOD1(G93A) transgenic mouse; and/or 2) the up-regulated transcripts may contain portions of 18S or 28S rRNA sequence. RT-PCRs were performed using poly(A)+ RNA as the template and oligo(dT)24-T7 as the primer for RT. Primers used for PCR are indicated in Fig. 1A : primer B and Q1 for 18S and primer E and Q1 for 28S. If there were rRNAs with poly(A) tails present in SOD1 mouse, we expected to obtain PCR products having complete 3' ends from the PCR primers (primer B or E) sites followed by poly(A) tail. We did obtain PCR products for both 18S and 28S (indicated as arrows in Fig. 1B ), and these PCR products were up-regulated in the SOD1(G93A) transgenic mouse. Sequencing of these PCR products revealed that they contained portions of rRNA sequence from primer sites followed by unknown sequence regions (USR) and poly(A) as schematically demonstrated in Fig. 1B . For 18S, the rRNA sequence ends at +1700 followed by 31 bp USR (GenBank accession no. AY248756); for 28S, the rRNA sequence ends at +4268 followed by 470 bp USR (GenBank accession no. AY248755). Both unknown sequences contained polyadenylation signals before the poly(A) (Fig. 1C , double underline). These results suggested that the up-regulated RNAs contain portions of 18S or 28S rRNA sequence.

To examine whether the transcripts corresponding to the identified PCR products contained the RDA fragment regions (clone 18S, 28S-1, or 28S-2), we performed PCRs using the same RT products as templates. A PCR product of the expected 1318 bp size was obtained when using primers A (corresponding to clone 18S) and F (corresponding to 18S USR), indicating that the corresponding transcript contained the clone 18S region. Likewise, a PCR product of the expected 750 bp size was obtained when using primers D (corresponding to clone 28S-2) and G (corresponding to 28S USR), but no product was obtained when using primers C (corresponding to clone 28S-1) and G, indicating that the corresponding transcript contained only the clone 28S-2 region. The clone 28S-1 may correspond to a different transcript.

To obtain full-length transcripts, we performed the 5' RLM-RACE (RNA ligase-mediated rapid amplification of cDNA end). Only authentic capped 5' ends of transcript are detected by RLM-RACE. We determined that the transcript corresponding to the identified clone 18S is a capped and polyadenylated transcript with virtual 100% homology to the 18S rRNA from +1 to +1700, followed by a 31 bp 3' terminal end containing a polyadenylation signal (Fig. 1C ). We named this transcript MSUR1 [mutant SOD1(G93A)-up-regulated RNA 1]. We were not able to determine the full-length transcript corresponding to 28S-2 by the presented approach. This is likely due to its larger sizes (4.5–5.5 kb) as determined by Northern blot analysis (see below).

The identified transcripts containing portions of rRNA are up-regulated in the spinal cord of SOD1(G93A) mouse
To confirm that the transcripts corresponding to the clones 18S, 28S-1, and 28S-2 were truly up-regulated in the spinal cord of the SOD1(G93A) mouse, we performed Northern blot analysis. Highly purified poly(A)+ RNA prepared from spinal cords of 2-month-old SOD1(G93A) mice or nontransgenic siblings were analyzed initially using RNA probes prepared from clones 18S, 28S-1, or 28S-2. As shown in Fig. 2 A, the 18S probe hybridized to a major 1.8 kb band, the 28S-1 probe hybridized to a 5 kb band and many bands of smaller size, and the 28S-2 probe hybridized to a 5 kb band and also many bands of smaller size. The 18S 1.8 kb band, 28S-1.5 kb band, and 28S-2.5 kb band as well as some of the smaller bands were up-regulated in the SOD1(G93A) spinal cord as compared to the nontransgenic spinal cord. This up-regulation was not observed in unaffected tissues, such as the kidney and brain, or in the spinal cords of transgenic mice overexpressing wild-type human SOD1 (data not shown). The equal loading of poly(A)+ RNA samples was confirmed by probing with a β-actin probe (Fig. 2A ). Furthermore, the β-actin probed blot showed a well-defined single band, indicating that the multiple bands were not degradation products. The specificity of the 18S, 28S-1, and 28S-2 signals was confirmed by their presence even after very high stringency washing as well as by virtue of their being greatly diminished on blots prehybridized with unlabeled antisense RNA prepared from the clones (Fig. 2A, MB ). The observed signals were not the result of incomplete 18S or 28S rRNA removal during the poly(A) selection, since the signal intensities of the probes derived from the different segments of 28S rRNA were not equal. Additionally, the banding patterns from the two probes were different, thereby suggesting that the observed signals were not the result of rRNA contamination of the poly(A)+ RNA pool. We further probed the blots with RNA probes prepared from the USR. As shown in Fig. 2B , the 18S-USR probe hybridized to a 1.7–1.8 kb band and the 28S-2-USR probe hybridized to a 4.5–5.0 kb band. These bands were up-regulated in the SOD1(G93A) spinal cord, as compared to the nontransgenic spinal cord. Densitometric analysis showed that transcripts corresponding to 18S, 28S-1, and 28S-2 were increased ~2.3-, 2.0-, and 2.1-fold, respectively, in the SOD1(G93A) spinal cord (Fig. 2C ).


Figure 2
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  		Figure 2. Northern blot analyses indicate that polyadenylated RNAs containing portions of rRNA are up-regulated in the spinal cord of SOD1(G93A) mouse. Each lane was equally loaded with 0.5 µg of poly(A)+ RNA prepared from 8-wk-old SOD1(G93A) or nontransgenic spinal cords. The blots were probed with RNA probes that were prepared from clones 18S, 28S-1, or 28S-2 (A). Blots were also probed with RNA probes that were prepared from the unknown sequence regions (B). A total of 6 SOD1 transgenic and 6 nontransgenic sibling mice were examined in 3 independent experiments with consistent results. C) Densitometric analysis of Northern blot results. *P < 0.001

The above results led us to speculate that there may be an overlooked group of capped and polyadenylated transcripts containing large portions of rRNA sequence. We searched the mouse EST database of GenBank using full sequences of both 18S and 28S rRNA (GenBank accession nos. X00686 and X00525, respectively). The initial search found >500 clones containing portions of 18S rRNA and >1000 clones, which contain portions of 28S rRNA. Further analysis revealed that these types of transcripts are not only widely expressed in tissues from many different organs, but are also expressed at multiple development stages and in diverse organisms.

MSUR1 rescues SOD1(G93A)-mediated cell death in HEK293 cells as well as in NSC34 cells
To explore the possible function of the identified transcript MSUR1, we transiently transfected HEK293 cells with different amounts of pcDNA3/MSUR1 but the same amount of pcDNA3/SOD1(G93A). We observed that cells transfected with pcDNA3/SOD1(G93A) alone started to detach from the dish at ~48 h after transfection. By 72 h post-transfection, a large number of transfected cells were detached. Trypan blue staining of these floating cells indicated that they were either dead or dying. This phenomenon was not observed in the cells transfected with pcDNA3/wild-type SOD1. Significantly, MSUR1 prevented SOD1(G93A)-mediated cell death in a dose-dependent manner (Fig. 3 A, B). Western blot analysis showed that the expression of SOD1(G93A) protein was the same in all samples (Fig. 3B ), indicating this protective effect was specifically due to MSUR1 expression.


Figure 3
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  		Figure 3. MSUR1 reduces SOD1(G93A)-mediated toxicity and cell death in HEK293 cells as well as in NSC34 cells. HEK293 cells were transiently transfected with indicated plasmid DNAs and then harvested for analysis at 72 h post-transfection. A) Bright-field microscopy of transfected cells showed that MSUR1 had a protective effect on SOD1(G93A)-induced cell death. B) Number of viable cells (assessed with cellular protein concentrations) was increased with increasing amount of MSUR1 cDNA transfected into cells (*P<0.002; **P<0.0001). Ratio of attached cells to dead cells was normalized to the cellular protein concentration of luciferase cDNA-transfected cells. Western blot analysis showed that the expression of SOD1(G93A) protein was the same in all samples. C) RT-PCR analysis of MSUR1 level in HEK293 stable lines showed that Clone F expresses more MSUR1 than Clone C. D) Indicated cell lines were transiently transfected with 1 µg of pCDNA/SOD1(G93A) and then harvested for determining cell viability at 72 h post-transfection. Cell viability was correlated with the level of MSUR1 expression (*P<0.01; **P<0.001). E) Hydroxyl radical levels were examined at 48 h post-transfection with pcDNA3/SOD1(G93A) by DCF assay. Hydroxyl radical level was reversely correlated with level of MSUR1 expression (*P<0.005; **P<0.0001). F) Protein oxidation levels were examined at 48 h post-transfection with pcDNA3/SOD1(G93A) by measuring protein carbonyl contents. Protein oxidation level was reversely correlated with the level of MSUR1 expression. Equal loading was confirmed by Ponceau S staining and by probe with anti-β-actin antibodies. G) NSC34 stable lines were transiently transfected with indicated plasmid DNAs and then harvested for determining cell viability at 72 h post-transfection. MSUR1 had a protective effect on SOD1(G93A)-induced cell death in NSC34 cells (*P<0.001).

To further confirm the observed protective effect, we generated HEK293 cell lines stably expressing MSUR1. Seven individual cell lines were established. The levels of MSUR1 mRNA were determined for each individual cell line by quantitative RT-PCR. Human SOD1 mRNA was used as an internal control. Clones C and F, which expressed the lowest and the highest MSUR1 mRNA levels, respectively, were selected for further analysis (Fig. 3C ). We transiently transfected Clones C, Clone F, and HEK293 cells with the same amount of pcDNA3/SOD1(G93A) and then measured cell viability at 72 h post-transfection. Clone F cells, which expressed more MSUR1, were more resistant to SOD1(G93A)-mediated cell death (Fig. 3D ). The cell viability was correlated with the level of MSUR1 expression. These results were consistent with the coexpression experiments (Fig. 3B ).

Increases in free radicals have been reported in tissues from transgenic mice expressing the catalytically active mutant SOD1(G93A), which is considered to be one of the toxicities of mutant SOD1(G93A) (21 , 22) . We examined free radical levels for each of the cell lines transfected with pcDNA3/SOD1(G93A) at 48 h post-transfection by DCF assay. The nonfluorescent fluorescin derivative [dichlorofluorescin (DCFH)], after being oxidized by free radicals, becomes DCF and emits fluorescence. Clone F cells had a significantly lower free radical level than Clone C or HEK293 cells (Fig. 3E ). Moreover, we measured protein carbonyl contents for each of the samples to determine the levels of protein oxidation. Clone F cells had significantly less protein oxidation than Clone C or HEK293 cells (Fig. 3F ). These results indicated that MSUR1 was able to reduce SOD1(G93A)-mediated free radical levels and oxidative damage.

NSC34 cells are hybrid mouse motor neuron/neuroblastoma cells that retain the ability to proliferate while exhibiting many motor neuron characteristics (23 24 25) . To examine whether the observed protective effect occurs in NSC34 cells, we generated NSC34-MSUR1 stable cell lines and also NSC34-luciferase stable cell lines as a control. Consistent with the observation in HEK293 cells, NSC34-MSUR1 cells were more resistant to SOD1(G93A)-mediated cell death when compared to NSC34-luciferase cells (Fig. 3G ). These results indicated that MSUR1 was able to reduce SOD1(G93A)-mediated toxicity and the consequent cell death.

MSUR1 reduces hydrogen peroxide-induced cytotoxicity in HEK293 cells as well as in NSC34 cells
We investigated whether MSUR1 has protective activity against the cytotoxicity produced by exposure to hydrogen peroxide (H2O2) in HEK293 cells. H2O2 has been frequently used in cell culture models to induce oxidative stress, as it is capable of altering the intracellular redox state by converting itself to highly reactive hydroxyl radicals (26 , 27) . HEK293, Clone C, and Clone F cells were treated with H2O2 for 30 min and then examined for free radical levels by DCF assay, as well as for oxidized protein levels by measuring protein carbonyl content. Clone F cells, which expressed more MSUR1, had the least free radical and protein oxidation levels among the three cell lines (Fig. 4 A, B). Furthermore, MTT assay was performed to measure mitochondria activity for these three cell lines after H2O2 treatment. The results showed that after H2O2 treatment, Clone F cells had significantly higher mitochondrial function as compared to Clone C or HEK293 cells (Fig. 4C ). We also performed H2O2 treatment experiments on NSC34 cells. Similar results were obtained in which NSC34-MSUR1 cells had a significantly lower free radical level (Fig. 4D ) and higher mitochondrial function (Fig. 4E ), as compared to NSC34-luciferase cells. These results indicated that MSUR1 was able to reduce H2O2-induced cytotoxicity.


Figure 4
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  		Figure 4. MSUR1 reduces hydrogen peroxide-induced cytotoxicity in HEK293 cells as well as in NSC34 cells. A) HEK293 stable lines were treated with indicated concentration of H2O2 for 30 min, and the hydroxyl radical levels were evaluated by DCF assay. Clone F cells had significantly less hydroxyl radical level than Clone C or HEK293 cells. B) Protein oxidation levels were examined at 3 h after 700 µM H2O2 treatment by measuring protein carbonyl contents. Clone F cells had significantly less protein oxidation compared to Clone C or HEK293 cells. Equal loading was confirmed by Ponceau S staining and by probe with anti-β-actin antibodies. C) Mitochondrial functions were measured by MTT assay at 3 h after 700 µM H2O2 treatment. Clone F cells had significantly higher mitochondrial function compared to Clone C or HEK293 cells (*P<0.01; **P<0.001). D) NSC34 stable lines were treated with indicated concentration of H2O2 for 30 min, and the hydroxyl radical levels were evaluated by DCF assay. NSC34-MSUR1 cells had significantly less hydroxyl radical level than NSC34-luciferase cells. E) Mitochondrial functions were measured by MTT assay at 3 h after H2O2 treatment. NSC34-MSUR1 cells had significantly higher mitochondrial function compared to NSC34-luciferase cells (*P<0.001).

MSUR1 does not encode a protein, suggesting a functional noncoding RNA
MSUR1 is an unusual RNA. It has a typical mRNA structure with a cap at the 5' end and poly(A) at 3' end but is, essentially, a portion of 18S rRNA. We investigated whether MSUR1 encodes a protein accounting for the observed protective effect. There are eight potential open reading frames (ORFs 1–8) presented on MSUR1 cDNA sequence (Fig. 5 A). To determine which ORF could encode MSUR1 protein, we prepared two deletion constructs: deletion 1, which deleted +1 to +591, resulting in missing ORF1–3; and deletion 2, which deleted +1 to +794, resulting in missing ORF1–3 and part of ORF4 (Fig. 5A ). Each construct was then cotransfected with pcDNA3/SOD1(G93A) into HEK293 cells, and the protective function was assessed by measuring cell survival and protein oxidation levels. The results showed that deletion 1 still had a protective function equivalent to full-length MSUR1 but that deletion 2 almost entirely lost protective function (Fig. 5B, C ). To further confirm the results, we generated NSC34-deletion 1 and NSC34-deletion 2 stable lines. These cell lines were treated with H2O2 and then measured for free radical level by DCF assay. NSC34-deletion 1 cells had a significantly lower free radical level compared to NSC34-luciferase cells, whereas NSC34-deletion 2 cells had only a slightly lower free radical level compared to NSC34-luciferase cells (Fig. 5D ). The difference between deletion 1 construct and deletion 2 construct is that the latter one destroyed ORF4, suggesting that ORF4 might be the one encoding for MSUR1 protein. However, the fact that the deletion 2 construct did not completely lose protective function suggested that ORF5 could not be ruled out.


Figure 5
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  		Figure 5. MSUR1 does not encode a protein, suggesting a functional noncoding RNA. A) Positions of 8 potential ORF and structures of 2 deletion constructs. B, C) HEK293 cells were transiently transfected with indicated plasmid DNAs and then harvested for determining cell viability at 72 h post-transfection (B) and for measuring protein oxidation level at 48 h post-transfection (C). Deletion 1 had a protective function as full length MSUR1, but deletion 2 almost entirely lost protective function (*P<0.0002). D) NSC34 stable lines were treated with indicated concentration of H2O2 for 30 min, and the hydroxyl radical levels were evaluated by DCF assay. NSC34-deletion 1 cells had significantly less hydroxyl radical level than NSC34-deletion 2 or NSC34-luciferase cells (*P<0.0001). E) In vitro translation showed that MSUR1 or deletion 1 did not produce any detectable protein product, whereas the control SMN cDNA produced the expected protein product. F) A stop codon was introduced at the second and the third amino acids of ORF4 (ORF4M) and ORF5 (ORF5M), respectively. Each mutant cDNA was then cotransfected with pcDNA3/SOD1(G93A) into HEK293 cells and determined protein oxidation levels. Both mutants still had protective function as full length MSUR1. Equal loading was confirmed by Ponceau S staining and by probe with anti-β-actin antibodies.

We performed in vitro translation to further assess whether MSUR1 encodes a protein. This was carried out in a coupled transcription/translation rabbit reticulocyte lysate system, using pcDNA3/MSUR1 and pcDNA3/deletion 1 plasmid DNAs. The expected molecular masses were ~3.9 kDa for ORF4 and ~5.9 kDa for ORF5. Thus, the products were analyzed by acrylamide gel electrophoresis under conditions which are able to resolve peptides as small as 2.5 kDa. As shown in Fig. 5E , no product was detected in full-length MSUR1 or deletion 1 sample, but the positive controls, including SMN protein (32 kDa) and luciferase protein (75 kDa; not shown), were properly produced. These results suggested that MSUR1 might not encode a protein. To further confirm this, we carried out site-directed mutagenesis experiments. A stop codon was introduced at the second and the third amino acids of ORF4 and ORF5, respectively. Each mutant cDNA was then cotransfected with pcDNA3/SOD1(G93A) into HEK293 cells, and the protective function was assessed by measuring cell survival and protein oxidation level. The results showed that both mutants still had as much protective function as full length MSUR1 (Fig. 5F shows the result of protein oxidation). Taking these results together, we concluded that MSUR1 is a functional noncoding RNA.

MSUR1 is widely expressed in various tissues and up-regulated in response to mutant SOD1(G93A)-mediated toxicity but not to H2O2-, glutamate-, or Aβ-mediated toxicity
MSUR1 expression profile was examined in multiple tissues of 60-day-old wild-type mice, including the brain, spinal cord, heart, liver, spleen, lung, kidney, and muscle, by RT-PCR analysis. The results showed that MSUR1 is widely expressed (Fig. 6 A).


Figure 6
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  		Figure 6. MSUR1 is widely expressed in various tissues and up-regulated in response to mutant SOD1(G93A)-mediated toxicity. A) RT-PCR analysis of MSUR1 expression in indicated tissues showed that MSUR1 is widely expressed. B, C) Primary cortical dissociated cultures were treated with 100 µM H2O2,20 µM glutamate or 3.3 µM Aβ1–42 for 16 h or transfected with pcDNA3/SOD1(G93A) or pcDNA3/EGFP and harvested at 72 h post-transfection. MSUR1 levels were determined by quantitative RT-PCR analysis. There was no significant difference in MSUR1 levels among DMEM control, H2O2, glutamate, and Aβ1–42 treated cells. There was ~45% up-regulation of MSUR1 level in pcDNA3/SOD1(G93A) transfected cells compared to pcDNA3/EGFP transfected cells (*P<0.001). Aβ1–42 was preincubated at 37°C for 24 h prior to adding it to the cultures.

Next, we investigated whether MSUR1 is up-regulated in response to various toxicities mediated by mutant SOD1(G93A), H2O2, glutamate, or Aβ1–42. Primary cortical dissociate cultures were used in this study. The 7-day-old cultures were treated with 100 µM H2O2, 20 µM glutamate, or 3.3 µM Aβ1–42 for 16 h and harvested for analysis. For the mutant SOD1(G93A) study, the 2-day-old cultures were transfected with pcDNA3/SOD1(G93A) or pcDNA3/EGFP and harvested at 72 h post-transfection. MSUR1 levels were determined by quantitative RT-PCR analysis. As shown in Fig. 6B , there was no significant difference in MSUR1 levels among DMEM control, H2O2, glutamate, and Aβ1–42 treated cells. There was ~45% up-regulation of MSUR1 level in pcDNA3/SOD1(G93A) transfected cells compared to pcDNA3/EGFP-transfected cells (Fig. 6C ), which is consistent with the mouse study.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we utilized representational difference analysis to identify the transcripts that are up-regulated or newly induced in the presymptomatic stage of SOD1(G93A) mice. Unexpectedly, three of our identified clones were rRNA segments, and they were the predominant clones found at 4 and 8 wk of age. On further analysis of one of these clones, it was discovered that the rRNA segments correspond to capped and polyadenylated transcripts containing long segments of sequence with 100% homology to 18S rRNA (MSUR1; Fig. 1 ). There are hundreds of known mRNAs containing short (20–50 nucleotides) rRNA sequences, an example of which is mouse IgE-binding factor mRNA, which contains 13 nucleotides of 18S (GenBank accession no. M10062). Mauro et al. (28) have shown that rRNA-like sequences occur in a wide variety of primary transcripts. They hypothesized that these rRNA-like sequences may function as cis-regulatory elements that regulate translational efficiency by interacting with rRNA or ribosomal proteins. Only a few known mRNAs contain long rRNA sequences. For example, mouse heat shock 86 protein mRNA contains 229 nucleotides of 28S (GenBank accession no. J04633; ref. 27 ). However, mouse EST database analysis indicates that there is a high abundance of uncharacterized polyadenylated RNAs that contain several hundred bases of rRNA sequence. The reason why only a few these types of transcripts have been identified may be because they are easily disregarded as experimental artifacts resulting from rRNA contamination of the poly (A)+ RNA pool.

What are the origins of these types of transcripts? Some of these RNAs may derive from genes thaat contain long rRNA sequences. It is possible that some of them may derive from rRNA. Some rRNAs may process to become mature mRNAs, which may have specific functions or may encode functional proteins. For example, the Humanin mRNA is 100% homologous to 16S rRNA gene and encodes a short polypeptide (29) . Humanin has been shown to rescue neuronal cell death caused by multiple different types of familial Alzheimer’s disease genes (30) . The rat homologue of Humanin, named Rattin, also displays protective activity against excitotoxic neuronal death (31 , 32) . Maximov et al. (32) have suggested that Humanin mRNA may be transcribed directly from the 16S gene but translated in the cytosol. The origins of these types of transcripts remain to be elucidated. It is possible that some transcripts of this type may have functional significance for some neurodegenerative disease processes, which will be investigated in the future.

One important finding from this study is that the identified MSUR1 has protective effects on SOD1(G93A)-mediated cell death in both HEK293 cells and NSC34 cells (Fig. 3) , suggesting that MSUR1 may be a rescue factor that is up-regulated against SOD1(G93A)-induced cell damage during the presymptomatic stage of the disease process in mice. In vivo, mutant SOD1 expression in multiple cell types, including microglia, astrocytes, oligodendrocytes, and motor neurons, is necessary to cause motor neuron death. The up-regulation of MSUR1 in mice might be a response of one or more cell types. It is possible that MSUR1 might play an important role in preventing motor neuron degeneration at an early age of mice. It will be worthwhile to investigate if over-expression of MSUR1 would delay the disease onset and life span of SOD1(G93A) mice. In addition, whether MSUR1 is also up-regulated in other mutant SOD1 mice and whether MSUR1 has protective effects on other SOD1 mutants remain to be investigated.

We observed that SOD1(G93A)-induced free radicals were significantly reduced in the cells expressing MSUR1 (Fig. 3) . Further, MSUR1 could also reduce H2O2-induced cytotoxicity in both HEK293 cells and NSC34 cells (Fig. 4) . It appears that the function of MSUR1 might be involved in oxidative stress. The mechanisms underlying the observed protective effects by MSUR1 remain to be elucidated.

In vitro translation and site-directed mutagenesis analysis suggests that MSUR1 is a functional noncoding RNA (Fig. 5) . More and more noncoding RNAs (ncRNAs) have been found to have essential functions in many pathophysiology processes other than simple intermediates in between DNA transcription and protein synthesis. According to their size, they can be classified into microRNA (~20 bp), small RNA (~20–300 bp), and medium or large sized RNA (over ~300bp, up to or over 10,000). Functions of medium or large sized RNA includes gene silencing, gene transcription, DNA imprinting, RNA interference, tumor suppressor, and relation to stress or apoptosis (33 34 35) . For example, heat shock RNA-1 (HSR1) is a cotranscriptional factor activator, along with elongation factor eEF1A to activate heat shock transcription factor 1 (36) . Furthermore, several noncoding RNAs involved in different central nervous system diseases have been identified in the last decades. For example, Prion diseases are primarily caused by an infectious protein designated PrP(Sc). The conversion of the normal PrP(C) to PrP(Sc) is the central pathogenic event. Two studies demonstrated that Prion associated RNAs are required to stimulate efficient PrP(SC) production and cause the pathological aspects of the diseases (37 , 38) . In our case, our hypothesis for the possible protective function is that MUSR1 might bind to some specific transcriptional factors, or may act by a RNA-DNA interaction, thereby up-regulating some antioxidant gene expression, enhancing the cellular antioxidant capacity. It is also possible that MSUR1 might directly interact with mutant SOD1 to affect mutant SOD1 activity and toxicity. Another possibility is that MSUR1 might protect mitochondria from generating excess free radicals or preventing mutant SOD1 entering the intermembrane space. The mechanisms underlying the protective functions will be investigated in the future.

RT-PCR analysis showed that MSUR1 is widely expressed in various tissues (Fig. 6A ), indicating its protective function is not limited to cells in the central nervous system. Further, MSUR1 is up-regulated in primary cortical dissociated cultures transfected with SOD1(G93A) cDNAs (Fig. 6B ), which supports the observations in mice. However, we did not observe obvious induction in the cultures treated with H2O2, glutamate or Aβ1–42, suggesting that the induction may be specific to SOD1(G93A)-mediated toxicity.

In summary, we presented a class of polyadenylated RNA that contain large portions of 18S or 28S rRNA sequence. MSUR1 belongs to this family of transcripts. Importantly, we demonstrated that MSUR1 has protective functions against mutant SOD1(G93A) toxicity, which implicates MSUR1 as a potential therapeutic target for ALS.


   ACKNOWLEDGMENTS
 
This work was supported by the ALS Association and National Institutes of Health grants MH-059805 and AG-17317. We thank N. Cashman and R. Liu for providing NSC34 cells, D. Wang and V. Sanders for access to the microplate reader, A. Burghes for SMN cDNA and access to the gel dryer, and J. R. Van Brocklyn for vigorous discussions.

Received for publication July 30, 2007. Accepted for publication September 13, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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