Use of A Randomized Hybrid Ribozyme Library for Identification of Genes Involved in Muscle Differentiation

abbreviations used are: siRNA, small interfering RNA; Rz, ribozyme We have employed 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 (p19 ARF and p21 WAF1 ), FGFR-4, fibronectin, Prkg2, Pdk4, fem and 6 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 sex-determining family of proteins, caused accelerated myotube formation and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful to understand the molecular pathways of muscle differentiation but also to design molecular strategies for myopathological therapies.

Ribozymes (Rz) 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)(4)(5)(6)(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 last two decades (8-10).
Hammerhead ribozymes recognize the target gene sequence by recognition arms at its 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 pool of randomized ribozymes (library) expressed from phenotype. Isolation and sequence analysis of ribozymes from the selected cells depicting the phenotype of interest is performed. The putative target mRNAs for the ribozymes isolated from cells are then predicted by DNA database search.
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)(15)(16)(17)(18)(19)(20)(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)(23)(24). These helicase coupled hybrid ribozymes were far 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)(28)(29)(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.  (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 (p19 ARF and p21 WAF1 ) in muscle differentiation implicating the significance of cell cycle regulation, and (iii) a novel gene (fem1) function involved in muscle differentiation.

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 was used for RT-PCR. It was reverse transcribed using lower primer (5'-TTT TTT TTT TTT TTT TTT TTG

Construction of Gene Specific Ribozymes and siRNA Expression
Vectors -RNA polymerase III driven hammerhead ribozyme expression plasmids for p19 ARF and p21 WAF-1 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, U6 promoter vector was used (40). Target sites for siRNA were selected using an algorithm (http://www.igene-therapeutics.co.jp). Sequences of the Rz and siRNA sites chosen for different genes are listed in Table 2

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 infected with the randomized ribozyme library showed significant abrogation of myotube formation ( Fig. 2b and d). Undifferentiating cells that continued to divide formed small colonies against the background of differentiating cells that fused to form myotubes (Fig. 2b).
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 RT-PCR and the sequence of these ribozymes was obtained as shown in the scheme in Fig. 1C and described in Material and methods.

Candidate Genes for C2C12 Differentiation
Known 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  (50). In light of this information, our screening assay for functional genes is proved to be highly efficient, easy and informative. In light of these data, isolation of cell cycle regulators as functionally involved in muscle differentiation was justified.

Tumor Suppressors and Cell Cycle Regulator Genes -
We further tested the functional role of p19 ARF and p21 WAF1 by their specific knockdown using ribozymes (Rz) and siRNA. Target sites for p19 ARF and p21 WAF-1 are shown in Table 2. Efficacy of the ribozymes to knockdown their targets (p19 ARF and p21 WAF1 ) was examined by exogenous expression of epitope-tagged proteins and Western blotting with tag-specific antibodies. We found that all the five p19 ARF Rz constructs and the two p21 WAF1 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 p19 ARF -Rz transfected myoblasts and found that 2/5 ribozymes (#19-2 and #19-4) resulted in delayed tube formation (Fig. 3A).  Table 2). One of the p19 ARF siRNA (#19-440) and both of the p21 WAF1 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 p19 ARF and p21 WAF1 in myoblast differentiation. Overexpression of fem-3 could overcome the feminizing effect of tra-2 and caused 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 fem protein in muscle differentiation by its specific knockdown with siRNA. Target sites are shown in Table 2.
Cells were transfected with siRNAs and selected in puromycin following which equal number of vector and fem siRNA tranfected cells were plated. We found that fem-siRNAs improved the proliferation potential of myoblasts (Fig. 4A, cf. b and c with a). We next transfected cells with expression plasmid for fem1 and found that these cells show early tube formation as compared to the control cells (Fig. 4B, cf. 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, cf. 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 ribozyme based functional gene discovery system. Further studies on specific knockdown of these genes are warranted.

Novel Gene Functions for Muscle Differentiation
These studies will not only enhance our understanding on 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 (p19 ARF and p21 WAF1 ) and a sex-determining protein fem1 in muscle differentiation.
We thank M. Miyagishi for help in siRNA vector construction. This work was supported in part by a research grant from AIST, Japan.