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Originally published In Press as doi:10.1074/jbc.M407428200 on September 24, 2004

J. Biol. Chem., Vol. 279, Issue 49, 51622-51629, December 3, 2004
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Use of a Randomized Hybrid Ribozyme Library for Identification of Genes Involved in Muscle Differentiation*

Renu Wadhwa{ddagger}, Tomoko Yaguchi{ddagger}, Kamaljit Kaur{ddagger}, Eigo Suyama{ddagger}, Hiroyuki Kawasaki§, Kazunari Taira{ddagger}§, and Sunil C. Kaul{ddagger}

From the {ddagger}Gene Function Research Center, National Institute of Advanced Industrial Science & Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan and the §Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan

Received for publication, July 2, 2004 , and in revised form, September 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ribozymes (Rz)1 are small RNA molecules that catalyze the hydrolysis of specific phosphodiester bonds of RNA strands with which they form base pairing and act as specific molecular scissors providing a very useful tool of studying gene function in vitro and in vivo (1, 2). Hammerhead ribozymes (HH-Rz) are among the smallest catalytic RNAs that have been used widely in molecular biology, biotechnology, and biomedicine (3-7). These RNAs fold into their active conformation by the binding of metal ions and cleave oligoribonucleotides at specific sites (NUX, where N can be any nucleotide and X can be A, C, or U) by mechanisms that have been widely studied in the last two decades (8-10). Hammerhead ribozymes recognize the target gene sequence by recognition arms at the 5'- and 3'-ends of its catalytic core. Thus for making a gene-specific ribozyme, its recognition arms (7-9 nucleotides each) are designed to include sequence complementary to the target mRNA. Randomization of these 7-9 nucleotides in each arm yields a large variety of ribozymes capable of targeting multiple mRNA substrates. Such a pool of randomized ribozymes (library) expressed from a pol III promoter were generated and used as an efficient gene discovery system (11-13). In contrast to the DNA microarrays and yeast two-hybrid systems, the ribozyme-based screening system has the potential to isolate genes directly involved in the phenomenon of interest. In this system, a randomized ribozyme library is expressed into the cells that are screened for a loss or gain of a biological phenotype. Isolation and sequence analysis of ribozymes from selected cells depicting the phenotype of interest was performed. The putative target mRNAs for the ribozymes isolated from cells were then predicted by DNA data base searches.

Successful inactivation of a specific gene in vivo by ribozymes depends on the appropriate design of the expression vector, level of expression, and its subcellular localization. Various improvements in designing ribozymes and their level of expression have been made in this regard (14-21). Besides target gene accessibility that plays a major role has been improved by designing the hybrid ribozymes that coupled the cleavage activity of hammerhead ribozymes with the unwinding activity of RNA helicase (22-24). These helicase-coupled hybrid ribozymes were far more effective in the cleavage of target mRNAs than their conventional counterparts (25, 26). Furthermore, libraries of hybrid ribozymes with randomized binding arms were predicted to have enhanced efficiency for rapid isolation of functional genes. Indeed, these were successfully used for isolation of genes involved in apoptosis (25, 27-30), cell migration and invasion (31, 32), and Alzheimer's disease (33). In the present study, we have used this novel, simple, efficient, and powerful method for isolation of genes involved in muscle differentiation in an in vitro cellular model system.

Skeletal muscle differentiation is characterized by muscle-specific gene expression, terminal withdrawal of cells from the cell cycle, their fusion into multinucleated cells, and assembly of the contractile apparatus (34). A number of studies in the recent past has unraveled the role of myogenic transcription factors, chromatin-modifying enzymes in initiation, and regulation of muscle differentiation (35, 36). Characterization of signaling cascades and gene functions involved in muscle differentiation is extremely valuable for understanding the biology of muscle disorders including myopathies, muscular dystrophy, and spinal muscular atrophy that involve deregulation of muscle differentiation (37). Mouse myoblast cell line, C2C12, provides an easy and convenient system to study myocyte differentiation. These cells can be differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis (38). We were able to interrupt differentiation of C2C12 cells by introduction of a randomized ribozyme library. Functional involvement of the predicted targets was confirmed by specific knockdown of genes by ribozymes or siRNA.

We demonstrate (i) the isolation of key regulators of muscle differentiation by the Rz-mediated functional gene discovery system validating the worth of this gene discovery system, (ii) the functional involvement of the tumor suppressor genes (p19ARF and p21WAF1) in muscle differentiation implicating the significance of cell cycle regulation, and (iii) a novel gene (fem1) function involved in muscle differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Construction of Randomized Hammerhead Ribozyme Libraries—Chemically synthesized oligonucleotides encoding ribozyme sequences with randomized substrate binding arms and a pol III termination sequence were converted to double-stranded sequences by PCR as described previously (27). After digestion with Csp45I and KpnI, the fragments were cloned downstream of the tRNA promoter in pUC-dt, and ribozymes were transcribed in vitro using AmpliScribe T7 transcription kit (Epicenter Technologies, Madison, WI) (25, 27). To generate poly(A)-connected Rz expression vectors, we inserted a poly(A) sequence of 60 nucleotides between the ribozyme and pol III termination sequence (Fig. 1, A and B). In the case of Rz expression retroviral vectors, ribozyme libraries were inserted into EcoRI and BamH I sites in the pMX-puro vector (25).



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  		FIG. 1.
Schematic presentation of randomized Rz-based gene discovery approach. A, cleavage of the target mRNA with HH-Rz. B, HH-Rz with randomized arms cloned into a retroviral expression vectors driven by a pol III promoter. Poly(A)-linked HH-Rz was embedded in tRNA and terminator. Ampicillin and puromycin resistance markers were used for selection in bacteria and mammalian cells, respectively. Abrogation of myoblast differentiation by randomized ribozyme library, rescue of ribozymes from undifferentiating cells, Rz cloning, sequencing, and target search by BLAST analysis (C) are shown.

 
Cell Culture, Infection, and Screening for Functional Genes—Mouse myoblasts C2C12 were used for assay. These were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin and fungizone (Invitrogen Life Technologies, Inc.) at 37 °C in an atmosphere of 5% CO2 and 95% air in a humidified incubator. Cells were induced to undergo muscle differentiation by culturing in the medium supplemented with 2% horse serum. pMX-puro/Rz library (8-10 µg of DNA per 10-cm dish) was transfected into mouse packaging cells, PLAT-E, using FuGENE 6 (Roche Applied Science). Cells were incubated with the DNA mixture overnight at 37 °C and then transferred at 32 °C for 48 h in normal Dulbecco's modified Eagle's medium to allow the packaging of DNA into the infectious viral particles. Culture medium was collected, filtered, and used as viral solution after an addition of polybrene (8 µg/ml). C2C12 cells were infected for 16 h followed by selection in puromycin (2 µg/ml)-supplemented medium for 24-48 h. Selected cells were then induced to differentiate. Undifferentiating and dividing cells were isolated from the differentiating nondividing cells by ring isolation. These were expanded and re-subjected to differentiating medium in a second round of selection. Undifferentiating clones were similarly tested through the third round of differentiation. RNA was prepared from undifferentiating clones using Isogen (Invitrogen). Total RNA (2 µg) was used for reverse transcriptase-PCR. It was reverse-transcribed using lower primer (5'-TTT TTT TTT TTT TTT TTT TTG GTA C-3') and MMLV transcriptase (42 °C, 90 min). Reverse-transcribed product was subjected to PCR amplification using upper (5'-tcc ccg gtt cga aac cgg gca-3') and lower primers (94 °C-52 °C-72 °C/30 s each, 20 cycles). The amplified PCR product (~150 bp) was cloned into a TA cloning vector (Promega) and sequenced using the T7 primer.

Construction of Gene-specific Ribozymes and siRNA Expression Vectors—RNA polymerase III-driven hammerhead ribozyme expression plasmids for p19ARF and p21WAF1 were made as described (26, 39). The empty vector containing the tRNA sequence but without ribozyme was used as a negative control.

For construction of siRNA expression vectors, the U6 promoter vector was used (40). Target sites for siRNA were selected using an algorithm (www.igene-therapeutics.co.jp). Sequences of the Rz and siRNA sites chosen for different genes are listed in Table II. In a typical example, sense (5'-caccGttGCtCACTtCAAGAGAGgtgtgctgtccCTCTCTTGGAGTGGGCGGCtttttt-3') and antisense (5'-GCATaaaaaaGCCGCCCACTCCAAGAGAGggacagcacacCTCTCTTGaAGTGaGCaaC-3') oligos were made; bold letters represent the target site sequence. C to T and G to A mutations (shown by lowercase bold letters) were inserted in the sense strand only. 5 µl of 100 µM sense and antisense oligos for a target site were mixed and annealed in 100-150 mM NaCl in a final volume (20 µl) using thermal cycler (99 °C for 2 min; 72 °C to 4 °C slope in 2 h). Annealed oligos were diluted (1:200), and 2 µl was ligated to a BspM I cut and gel-purified pciPur vector (40) using high ligation kit (Takara). Plasmid DNA prepared from the transformed bacteria was sequenced with a vector primer (CAGGAAACAGCTATGAC) for confirmation of the integrity of the cloned DNA fragments.


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  		TABLE II
Target site sequences of ribozymes and siRNA constructs

 
Analysis of Functional Involvement of Genes during Muscle Differentiation—C2C12 cells were transfected with specific ribozyme or with siRNA gene knockdown constructs using LipofectAMINETM PLUS (Invitrogen). Transfected cells were selected in puromycin (2.5 µg/ml)-supplemented medium for 2-4 days and then subjected to differentiation. Vector-transfected cells were used as control. Myotube formation was monitored in control and in gene knockdown cells. Expression of muscle-specific genes in control and gene knockdown cells were analyzed by Western blotting with specific antibodies as given below.

Western Blot Analysis—Immunoassays were performed as described (26). Anti-myogenin (F5D, Santa Cruz), anti-p21WAF1 (C-19, Santa Cruz), anti-V5 (Invitrogen), and anti-actin (Chemicon) antibodies were used. The immunocomplexes formed were visualized with horseradish peroxidase-conjugated secondary antibodies using ECL PLUS kit (Amersham Biosciences).

Immunostaining—C2C12 cells and its derivatives (transfected with either expression or siRNA plasmids for fem1 as indicated) were induced for differentiation by incubating in the growth medium supplemented with 2% horse serum. Cells were fixed with methanol/acetone (1:1) at various time points and then stained with anti-MHC antibody (Novocastra). Alexa-488-conjugated goat anti-mouse (Molecular Probes) antibody was used as secondary antibody. The cells were examined on a Carl Zeiss microscope.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Abrogation of C2C12 Differentiation with Randomized Hybrid Ribozyme Library—To explore the genes functionally involved in muscle differentiation, C2C12 myoblasts were infected with randomized ribozyme library and subjected to differentiation medium as shown in Fig. 1C. Whereas control (empty vector-infected) cells formed full myotubes in 72-96 h in differentiation medium (Fig. 2, panels a and c), cells infected with the randomized ribozyme library showed significant abrogation of myotube formation (Fig. 2, panels b and d). Undifferentiating cells that continued to divide formed small colonies against the background of differentiating cells that fused to form myotubes (Fig. 2, panel b). We isolated 96 and 12 undifferentiating clones from the Rz- and empty vector-infected cultures, respectively (Fig. 2). Cells were expanded to 60-70% confluency in 48- and 24-well plates for second and third rounds of selection for undifferentiating clones. We obtained 67 undifferentiating clones from the Rz-infected culture and none from the empty vector-infected culture. Ribozymes were recovered from the undifferentiating cells by reverse transcriptase-PCR, and the sequence of these ribozymes was obtained as shown in the scheme in Fig. 1C and described under "Materials and Methods."



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  		FIG. 2.
Abrogation of C2C12 differentiation by randomized Rzs. C2C12 control (panel a) and randomized ribozyme library transduced (panel b) cells cultured in differentiation medium (panels c and d) are shown. Library transduced myoblasts did not form myotubes.

 
Candidate Genes for C2C12 Differentiation—Known key regulators of muscle differentiation by three rounds of selection, we isolated 67 independent undifferentiating C2C12 derivative clones. Ribozyme sequences recovered from these clones matched with 33 different target sites that aligned to 24 different mRNAs (Table I). Five (MyoD1, Mylk, myosin, myogenin, and Myf5) out of the twenty-four were the genes specifically expressed in muscles. Myf5 is the first gene to be expressed followed by myogenin, myoD, and myf6 during embryonic muscle development in mice. Its expression is detected in mononucleated myoblasts whereas myogenin and myoD accumulate in mono- and multinucleated myogenic cells (41). MyoD and Myf5 are master regulatory genes for myogenic determination, and myogenin and myosin (muscle structural proteins) are important for terminal differentiation and lineage maintenance (42, 43). Although Mylk (myosin light chain kinase), and myosin are known to be involved mainly in muscle contractility (44), our data suggest that they may also be involved in muscle differentiation. Based on the characteristics and known function of the isolated genes, their detection strongly validated the use of ribozyme-based functional gene discovery approach.


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  		TABLE I
Ribozyme sequences and their putative target genes

 
Genes Involved in Extracellular Signaling: Fibronectin and FGFR4 —Five ribozymes matched to three independent sites on fibronectin. Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation (45). Differences in fibronectin conformations were shown to alter the quantity of bound subunits of integrin and regulate differentiation. Treatment of myoblasts with specific inhibitors of proteoglycan synthesis (sodium chlorate and {beta}-D-xyloside) substantially affected the deposition and assembly of the extracellular matrix (ECM) constituents (glypican, fibronectin, and laminin) and meddled their differentiation without affecting the expression of muscle differentiation regulators (MyoD, MEF2A, and myogenin). Treatment of differentiated myoblast with RGDS peptides completely inhibited myogenesis without affecting myogenin expression. Interestingly, antibodies specific for the RGD binding site in fibronectin abolished myoblast differentiation (46-48). Based on these data, it was concluded that the expression of myogenin is not sufficient to successfully drive muscle formation and that ECM is required to complete the skeletal muscle differentiation process.

FGFs were identified as powerful stimulators of myoblast proliferation and inhibitors of myoblast differentiation in vitro. In chick embryos, most if not all, replicating myoblasts, but not the differentiated muscle cells, expressed high levels of the FGF receptor FREK/FGFR4. It preceded MyoD expression that signals the onset of terminal differentiation suggesting an important role of the FGF receptor in muscle differentiation and one of the earliest molecular markers (49). It was shown that an inhibition of FGFR4, but not FGFR1 signaling affects Myf5, MyoD, and myosin heavy chain expression and dramatic loss of limb muscles. Conversely, overexpression of FGF8 in somites promoted FGFR4 expression and muscle differentiation. FGFR4 signaling was denoted as a crucial step in the cascade of molecular events leading to terminal muscle differentiation (50). In light of this information, our screening assay for functional genes is proved to be highly efficient, easy and informative.

Tumor Suppressors and Cell Cycle Regulator Genes—Three of the twenty-four target genes were the tumor suppressors and cell cycle regulators (p27, p19ARF, and p21WAF1). During muscle differentiation, muscle-specific gene induction and transition of cells from the proliferative stage to form post-mitotic multinucleated myotubes are regulated through highly ordered and temporally separable events. Cell cycle arrest is critical for muscle differentiation and involves the inhibitors of cyclin-dependent kinases (51-53). The level of p27 protein gradually increases with differentiation (54). p19ARF is an upstream regulator of p53 and p21WAF1 (inhibitor of cyclin-dependent kinase). By inhibiting the cyclin-dependent kinase, p21WAF1 regulates the activity of retinoblastoma tumor suppressor protein (Rb); hypophosphorylated pRb binds to E2F and causes G0 cell cycle arrest. p21 expression is also activated by a muscle-specific transcriptional regulator, MyoD, independent to that of p53 and causes cell cycle withdrawal (55). p21-/- mice display increased cell number and enhanced cell cycle progression of myogenic progenitor cells and impaired muscle differentiation and regeneration (56, 57). Duchenne muscular dystrophy (DMD, decreased muscle cell proliferation phenotype), caused by the absence of dystrophin, involves an increased expression of p21WAF1. Interestingly, an appropriate transient transfection of p21-antisense oligos improved their proliferation (58). In light of these data, isolation of cell cycle regulators as functionally involved in muscle differentiation was justified.

We further tested the functional role of p19ARF and p21WAF1 by their specific knockdown using Rz and siRNA. Target sites for p19ARF and p21WAF1 are shown in Table II. Efficacy of the ribozymes to knockdown their targets (p19ARF and p21WAF1) was examined by exogenous expression of epitope-tagged proteins and Western blotting with tag-specific antibodies. We found that all the five p19ARF Rz constructs and the two p21WAF1 Rz constructs could bring down the expression of their respective targets by 30-50% (data not shown). We next traced the formation of C2C12 myotubes in control and p19ARF-Rz transfected myoblasts and found that 2 of 5 ribozymes (19-2 and 19-4) resulted in delayed tube formation (Fig. 3A). This was also accompanied by a decrease in myogenin; an established marker for myotube formation. The p19ARF Rz derivatives also showed a lower level of p21WAF1 and myogenin compared with the vector-transfected control cells. Of note, the effect of different p19ARF Rzs on p21WAF1 and myogenin was well correlated with their effect on differentiation potential; the Rzs that effectively reduced p21WAF1 and myogenin-constrained myoblast differentiation.



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  		FIG. 3.
Myotube formation in control C2C12 (19-0) and their p19ARF-Rz derivatives (19-1 to 5). Cells transfected with 19-2 and 19-4 showed delay in myotube formation (upper panel). These cells also showed decreased levels of p21WAF1 and myogenin (lower panel) (A). Myotube formation in C2C12 control (21-0) and p21WAF1-Rz-transfected cells is shown. Cells transfected with Rzs 21-1 and 21-4 showed delay in myotube formation (upper panel) and decreased amount of myogenin (lower panel) (B). Decreased levels of p19ARF and p21WAF1 expression in myoblasts transfected with p19ARF and p21WAF1 siRNAs are shown (C). Cells showing effective knockdown of p19ARF (19-440) and p21WAF1 (21-59 and 21-625) showed decreased myogenin (C) and delayed myotube formation (D).

 
We next targeted p21WAF1 with ribozymes. Interestingly, one of the two p21WAF1 ribozymes that caused sharp decrease in myogenin resulted in significant delay in myotube formation (Fig. 3B). Whereas control cells formed full myotubes in 60 h, the 21-4 Rz-transfected cells showed a high population of un-aligned myoblasts. A more efficient knockdown of p19ARF and p21WAF1 was attempted with siRNAs (target sites are shown in Table II). One of the p19ARF siRNA (19-440) and both of the p21WAF1 siRNA (21-59 and 21-625) (Fig. 3C) resulted in decreased expression of the respective target genes and myogenin. This was accompanied by significantly delayed myotube formation (Fig. 3D). These data confirmed the functional involvement of p19ARF and p21WAF1 in myoblast differentiation.

A Novel Function of Sex-determining Family Protein-fem—Caenorhabditis elegans Fem genes (fem1, fem2, and fem3) are centrally involved in male sexual development including initiation of spermatogenesis in XX (hermaphrodite) worms, and the entire spectrum of male differentiation in XO animals (59, 60). Fem proteins are conserved in mice and human, suggesting their function in similar pathways. These contain 6-7 contiguous copies of a motif (ANK repeats) found in cell cycle-regulating proteins (the cdc10 of Schizosaccharomyces pombe, the SWI6 gene of Saccharomyces cerevisiae, the Notch gene of Drosophila, and the lin-12 and glp-1 genes of C. elegans) (61). The Fem2 sequence is related to protein serine/threonine phosphatases of Type 2C (PP2C) and exhibits magnesium-dependent casein phosphatase activity that is critical for its function in male development in worms (62). Murine homologues fem1a and fem1b are expressed during embryogenesis; the fem1a expression is enriched in adult heart and skeletal muscle, and fem1b is highly expressed in adult testis suggesting their unique tissue-specific functions (63, 64). Fem proteins are negatively regulated by Tra-2, which prevents male development; a balance between the opposing activities of Tra-2A and Fem-3 determines sex-specific cell fates in somatic tissues. Overexpression of fem-3 could overcome the feminizing effect of Tra-2 and cause widespread masculinization of XX somatic tissues (65).

In our screen, seven ribozymes that hit three independent sites of murine Fem homolog (fem1c) were isolated. We decided to analyze the functional involvement of the Fem protein in muscle differentiation by its specific knockdown with siRNA. Target sites are shown in Table II. Cells were transfected with siRNAs and selected in puromycin after which equal numbers of vector and fem siRNA-transfected cells were plated. We found that Fem-siRNAs improved the proliferation potential of myoblasts (Fig. 4A, compare panels b and c with a). We next transfected cells with expression plasmid for fem1 and found that these cells showed early tube formation compared with the control cells (Fig. 4B, compare panels c and d with a and b). Transfection of fem1 siRNAs (Fem-1000 and Fem-2203) caused remarkable delay in tube formation as visualized by phase contrast microscopy (Fig. 4B, compare panels e and f with a and b) and immunostaining with anti-myosin heavy chain antibody (Fig. 4C). This was accompanied by decrease in myogenin level (Fig. 4D). In the absence of antibodies specific to Fem1, specificity of Fem-siRNAs to its target was ensured by exogenous expression of V5-tagged fem, and its analysis by anti-V5 antibodies. We found that these siRNAs were highly specific to Fem1 and caused up to 55% reduction in the protein level (Fig. 4E). Taken together, we have exposed a novel function of the Fem1 protein by employing a ribozyme-based functional gene discovery system.



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  		FIG. 4.
Effect of fem knockdown and overexpression on C2C12 differentiation. Increased proliferation of C2C12 myoblasts transfected with siRNA for fem1 (A). Cells transfected with fem1 expression plasmid showed enhanced and early myotube formation (B, compare panels c with a and b), and the cells transfected with expression plasmid and siRNA for fem1 showed delayed myotube formation (B, compare panels d with e and f). Myosin heavy chain staining of C2C12 cells and their derivatives (fem1-overexpressing and fem1-compromised) after 36 and 72 h of induction of differentiation (C) is shown. Cells transfected with siRNA for fem1 showed decreased myogenin (D). Cells transfected with siRNA for Fem (1000 and 2203) showed decreased amount of exogenous V5-tagged full-length Fem (1-616 amino acids) and its N terminus 400 amino acids (1-400). V5-tagged mortalin was used as a negative control, and no change in its expression level was seen (E).

 
Novel Gene Functions for Muscle Differentiation—Our screen has identified six novel genes (GenBankTM accession numbers: 1700025D19, 2410015A15, 4933405K18, 6820443O06, A630095P14, and A930024E05 (Table I) putatively involved in muscle differentiation. Further studies on specific knockdown of these genes are warranted. These studies will not only enhance our understanding of the functional biology of muscle differentiation but may also provide information for therapy of muscle diseases.

Taken together, we have demonstrated (i) an effective use of a randomized ribozyme library for identifying genes for muscle differentiation, (ii) the validity of this approach by specific knockdown of some mRNAs, and (iii) the functional involvement of tumor suppressors (p19ARF and p21WAF1) and a sex-determining protein Fem1 in muscle differentiation.


   FOOTNOTES
 
* This work was supported in part by a research grant from the National Institute of Advanced Industrial Science & Technology (AIST). 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. Back

To whom correspondence should be addressed: Gene Function Research Center, AIST, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. Tel.: 81-29-861-6713; Fax: 81-29-861-2900; E-mail: s-kaul{at}aist.go.jp.

1 The abbreviations used are: Rz, ribozyme; siRNA, small interfering RNA; pol, polymerase; ECM, extracellular matrix; FGF, fibroblast growth factor; Rb, retinoblastoma; HH-Rz, hammerhead ribozymes. Back


   ACKNOWLEDGMENTS
 
We thank M. Miyagishi for help in siRNA vector construction.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Uhlenbeck, O. C. (1987) Nature 328, 596-600[CrossRef][Medline] [Order article via Infotrieve]
  2. Cech, T. R. (2002) Biochem. Soc. Trans. 30, 1162-1166[CrossRef][Medline] [Order article via Infotrieve]
  3. Stage-Zimmermann, T. K., and Uhlenbeck, O. C. (1998) RNA 4, 875-889[CrossRef][Medline] [Order article via Infotrieve]
  4. Rossi, J. J. (1999) Curr. Opin. Mol. Ther. 1, 316-322[Medline] [Order article via Infotrieve]
  5. Michienzi, A., Castanotto, D., Lee, N., Li, S., Zaia, J. A., and Rossi, J. J. (2003) Ann. N. Acad. Sci. 1002, 63-71[CrossRef][Medline] [Order article via Infotrieve]
  6. Sioud, M. (2001) Curr. Mol. Med. 1, 575-588[CrossRef][Medline] [Order article via Infotrieve]
  7. Kato, Y., Kuwabara, T., Toda, H., Warashina, M., and Taira, K. (2000) Nucleic Acids Symp. Ser. 44, 283-284[Abstract/Free Full Text]
  8. Verma, S., Vaish, N. K., and Eckstein, F. (1997) Curr. Opin. Chem. Biol. 1, 532-536[CrossRef][Medline] [Order article via Infotrieve]
  9. Takagi, Y., Suyama, E., Kawasaki, H., Miyagishi, M., and Taira, K. (2002) Biochem. Soc. Trans. 30, 1145-1149[CrossRef][Medline] [Order article via Infotrieve]
  10. Goodchild, J. (2002) Exp. Opin. Ther. Targets 6, 235-247
  11. Kruger, M., Beger, C., Li, Q. X., Welch, P. J., Tritz, R., Leavitt, M., Barber, J. R., and Wong-Staal, F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8566-8571[Abstract/Free Full Text]
  12. Welch, P. J., Marcusson, E. G., Li, Q. X., Beger, C., Kruger, M., Zhou, C., Leavitt, M., Wong-Staal, F., and Barber, J. R. (2000) Genomics 66, 274-283[CrossRef][Medline] [Order article via Infotrieve]
  13. Kawasaki, H., and Taira, K. (2001) Nucleic Acids Res. Suppl. 1, 133-134
  14. Koseki, S., Tanabe, T., Tani, K., Asano, S., Shioda, T., Nagai, Y., Shimada, T., Ohkawa, J., and Taira, K. (1999) J. Virol. 73, 1868-1877[Abstract/Free Full Text]
  15. Kuwabara, T., Warashina, M., and Taira, K. (2002) J. Biochem. 132, 149-155[Abstract/Free Full Text]
  16. Miyagishi, M., Kuwabara, T., and Taira, K. (2001) Cancer Chemother. Pharmacol. 48, S96-101[Medline] [Order article via Infotrieve]
  17. Kuwabara, T., Warashina, M., and Taira, K. (2000) Trends Biotechnol. 18, 462-468[CrossRef][Medline] [Order article via Infotrieve]
  18. Sano, M., Kuwabara, T., Warashina, M., Fukamizu, A., and Taira, K. (2002) Antisense Nucleic Acid Drug Dev. 12, 341-352[CrossRef][Medline] [Order article via Infotrieve]
  19. Sano, M., Kuwabara, T., Nara, Y., Warashina, M., and Taira, K. (2000) Nucleic Acids Symp. Ser. 44, 203-204[Abstract/Free Full Text]
  20. Zhao, J. J., and Lemke, G. (1998) Mol. Cell Neurosci. 11, 92-97[CrossRef][Medline] [Order article via Infotrieve]
  21. Iyo, M., Kawasaki, H., and Taira, K. (2004) Methods Mol. Biol. 252, 257-265[Medline] [Order article via Infotrieve]
  22. Warashina, M., Kuwabara, T., Kato, Y., Sano, M., and Taira, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5572-5577[Abstract/Free Full Text]
  23. Kawasaki, H., Warashina, M., Kuwabara, T., and Taira, K. (2004) Methods Mol. Biol. 252, 237-243[Medline] [Order article via Infotrieve]
  24. Akashi, H., Kawasaki, H., Kim, W. J., Akaike, T., Taira, K., and Maruyama, A. (2002) J. Biochem. 131, 687-692[Abstract/Free Full Text]
  25. Kawasaki, H., and Taira, K. (2002) EMBO Rep. 3, 443-450[CrossRef][Medline] [Order article via Infotrieve]
  26. Wadhwa, R., Ando, H., Kawasaki, H., Taira, K., and Kaul, S. C. (2003) EMBO Rep. 4, 595-601[CrossRef][Medline] [Order article via Infotrieve]
  27. Kawasaki, H., Onuki, R., Suyama, E., and Taira, K. (2002) Nat. Biotechnol. 20, 376-380[CrossRef][Medline] [Order article via Infotrieve]
  28. Kawasaki, H., and Taira, K. (2002) Nucleic Acids Res. 30, 3609-3614[Abstract/Free Full Text]
  29. Kawasaki, H., Tsunemi, M., Iyo, M., Oshima, K., Minoshima, H., Hamada, A., Onuki, R., Suyama, E., and Taira, K. (2002) Nucleic Acids Res. Suppl. 2, 275-276
  30. Onuki, R., Kawasaki, H., Baba, T., and Taira, K. (2003) Antisense Nucleic Acid Drug Dev. 13, 75-82[CrossRef][Medline] [Order article via Infotrieve]
  31. Suyama, E., Kawasaki, H., Kasaoka, T., and Taira, K. (2003) Cancer Res. 63, 119-124[Abstract/Free Full Text]
  32. Suyama, E., Kawasaki, H., Nakajima, M., and Taira, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5616-5621[Abstract/Free Full Text]
  33. Onuki, R., Bando, Y., Suyama, E., Katayama, T., Kawasaki, H., Baba, T., Tohyama, M., and Taira, K. (2004) EMBO J. 23, 959-968[CrossRef][Medline] [Order article via Infotrieve]
  34. Taylor, M. V. (2003) Curr. Biol. 13, R964-R966[CrossRef][Medline] [Order article via Infotrieve]
  35. McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2002) Curr. Opin. Cell Biol. 14, 763-772[CrossRef][Medline] [Order article via Infotrieve]
  36. Pownall, M. E., Gustafsson, M. K., and Emerson, C. P., Jr. (2002) Annu. Rev. Cell Dev. Biol. 18, 747-783[CrossRef][Medline] [Order article via Infotrieve]
  37. Amack, J. D., and Mahadevan, M. S. (2004) Dev. Biol. 265, 294-301[CrossRef][Medline] [Order article via Infotrieve]
  38. Shen, X., Collier, J. M., Hlaing, M., Zhang, L., Delshad, E. H., Bristow, J., and Bernstein, H. S. (2003) Dev. Dyn. 226, 128-138[CrossRef][Medline] [Order article via Infotrieve]
  39. Kato, Y., Kuwabara, T., Warashina, M., Toda, H., and Taira, K. (2001) J. Biol. Chem. 276, 15378-15385[Abstract/Free Full Text]
  40. Miyagishi, M., and Taira, K. (2002) Nat. Biotechnol. 20, 497-500[CrossRef][Medline] [Order article via Infotrieve]
  41. Rohwedel, J., Maltsev, V., Bober, E., Arnold, H. H., Hescheler, J., and Wobus, A. M. (1994) Dev. Biol. 164, 87-101[CrossRef][Medline] [Order article via Infotrieve]
  42. Wei, Q., and Paterson, B. M. (2001) FEBS Lett. 490, 171-178[CrossRef][Medline] [Order article via Infotrieve]
  43. Franchini, M. (2003) Recent Prog. Med. 94, 478-483
  44. Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267[CrossRef][Medline] [Order article via Infotrieve]
  45. Bouzeghrane, F., Mercure, C., Reudelhuber, T. L., and Thibault, G. (2004) J. Mol. Cell Cardiol. 36, 343-353[CrossRef][Medline] [Order article via Infotrieve]
  46. Osses, N., and Brandan, E. (2002) Am. J. Physiol. Cell Physiol. 282, C383-C394[Abstract/Free Full Text]
  47. Morla, A. O., and Mogford, J. E. (2000) Biochem. Biophys. Res. Commun. 272, 298-302[CrossRef][Medline] [Order article via Infotrieve]
  48. Garcia, A. J., Vega, M. D., and Boettiger, D. (1999) Mol. Biol. Cell 10, 785-798[Abstract/Free Full Text]
  49. Marcelle, C., Wolf, J., and Bronner-Fraser, M. (1995) Dev. Biol. 172, 100-114[CrossRef][Medline] [Order article via Infotrieve]
  50. Marics, I., Padilla, F., Guillemot, J. F., Scaal, M., and Marcelle, C. (2002) Development 129, 4559-4569[Abstract/Free Full Text]
  51. Andres, V., and Walsh, K. (1996) J. Cell Biol. 132, 657-666[Abstract/Free Full Text]
  52. Adams, G. R., Haddad, F., and Baldwin, K. M. (1999) J. Appl. Physiol. 87, 1705-1712[Abstract/Free Full Text]
  53. Cabane, C., Englaro, W., Yeow, K., Ragno, M., and Derijard, B. (2003) Am. J. Physiol. Cell Physiol. 284, C658-666[Abstract/Free Full Text]
  54. Franklin, D. S., and Xiong, Y. (1996) Mol. Biol. Cell 7, 1587-1599[Abstract]
  55. Halevy, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J., Hannon, G. J., Beach, D., and Lassar, A. B. (1995) Science 267, 1018-1021[Abstract/Free Full Text]
  56. Hawke, T. J., Meeson, A. P., Jiang, N., Graham, S., Hutcheson, K., DiMaio, J. M., and Garry, D. J. (2003) Am. J. Physiol. Cell Physiol. 285, 25
  57. de la Serna, I. L., Roy, K., Carlson, K. A., and Imbalzano, A. N. (2001) J. Biol. Chem. 276, 41486-41491[Abstract/Free Full Text]
  58. Endesfelder, S., Bucher, S., Kliche, A., Reszka, R., and Speer, A. (2003) J. Mol. Med. 81, 355-362[CrossRef][Medline] [Order article via Infotrieve]
  59. Doniach, T., and Hodgkin, J. (1984) Dev. Biol. 106, 223-235[CrossRef][Medline] [Order article via Infotrieve]
  60. Kimble, J., Edgar, L., and Hirsh, D. (1984) Dev. Biol. 105, 234-239[CrossRef][Medline] [Order article via Infotrieve]
  61. Spence, A. M., Coulson, A., and Hodgkin, J. (1990) Cell 60, 981-990[CrossRef][Medline] [Order article via Infotrieve]
  62. Chin-Sang, I. D., and Spence, A. M. (1996) Genes Dev. 10, 2314-2325[Abstract/Free Full Text]
  63. Ventura-Holman, T., and Maher, J. F. (2000) Biochem. Biophys. Res. Commun. 267, 317-320[CrossRef][Medline] [Order article via Infotrieve]
  64. Ventura-Holman, T., Lu, D., Si, X., Izevbigie, E. B., and Maher, J. F. (2003) Gene (Amst.) 314, 133-139[CrossRef][Medline] [Order article via Infotrieve]
  65. Mehra, A., Gaudet, J., Heck, L., Kuwabara, P. E., and Spence, A. M. (1999) Genes Dev. 13, 1453-1463[Abstract/Free Full Text]

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