A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation.
As a tool for functional genomics, a hairpin ribozyme gene library with randomized target recognition sequences was constructed in a retroviral vector. This library has the potential to target and cleave any possible RNA substrate. Mouse fibroblasts transduced with this ribozyme gene vector library were selected in a focus formation assay to isolate in vivo functional ribozymes that promote cell transformation in tissue culture. After two successive rounds of selection by focus formation assay, a transforming ribozyme (Rz007) was identified. The sequence of this ribozyme was used to identify the putative target genes responsible for the transformation. A candidate gene target for Rz007 encodes telomerase reverse transcriptase (mTERT). Both mRNA level and enzymatic activity of mTERT were down-regulated in Rz007-transformed cells. Furthermore, newly designed ribozymes, recognizing other potential ribozyme cleavage sites unique to the mTERT mRNA, also cause cell transformation, thus validating the role of mTERT in suppressing the transformation phenotype. These surprising results suggest that the commonly accepted role of telomerase in maintaining cellular immortalization is more complicated than previously thought. These studies also demonstrate the utility of this novel 'reverse' functional genomics approach, enabling the targeted discovery of genes, whether previously known or not, that are involved in any selectable phenotype.
Li
Q X
QX
Immusol Inc., San Diego, CA 92121, USA.
Robbins
J M
JM
Welch
P J
PJ
Wong-Staal
F
F
Barber
J R
JR
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D048429:Cell Size; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012313:RNA; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014410:Tumor Stem Cell Assay
D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase