Factors affecting biosynthesis of BC200 RNA

Abstract

RNA polymerase III is one of eukaryotic polymerases and transcribes tRNA, 5S rRNA, and many other noncoding RNAs. Generally, to recruit RNA polymerase III into promoters of the noncoding RNA genes, various transcription factors including TFIIIC and TFIIIB were needed. Especially, TFIIIB is a target regulator affected by some oncogenes or tumor suppressor genes. Therefore, non-coding RNAs transcribed by RNA polymerase III can be overexpressed in tumor cells. Brain cytoplasmic 200 RNA (BC200 RNA), which was first identified as a noncoding RNA in neuronal cells, transcribed by RNA polymerase III. Since BC200 RNA was not observed in somatic cells other than neurons, it was regarded as a neural-specific RNA. Howev-er, BC200 RNA is also highly expressed in a number of tumors that are of non-neuronal origin. Therefore, it is conceivable that there exists a regulatory circuit for BC200 RNA expression in tumor cells. Since promoter elements for BC200 RNA transcription have not been identified yet, in this thesis, the promoter elements re-quired for efficient expression of BC200 RNA were analyzed in HeLa cervical carcinoma cells. Mutagenesis analysis showed that internal A box, positioned at +5 to +15 and B box, positioned at +78 to +88 relative to the 5’ end of BC200 RNA are crucial for BC200 RNA expression. To assess the upstream promoter of BC200 RNA gene, deletion analysis from the upstream -1010 sequence was performed. The deletion of the upstream sequence to -100 did not significantly affect the BC200 transcription, but further deletion de-creased the transcription. The promoter gradually lost its transcriptional efficiency as the deletion proceeded. When the whole upstream sequence was deleted, the transcription dropped to less than 1%. This result sug-gests that the upstream -100 sequence is essential for efficient transcription of BC200 RNA and that there might be many transcription factors binding to this upstream region. Mutational analysis of putative protein binding sites demonstrated that the -64 to -59, the -28 to -22, and -17 to -12 regions are more effective sites for efficient transcription. The effectiveness of these sites was also confirmed by competition assays. The putative binding proteins for the three sites would be cETS-2/MAF, TBP, and c-Myb, respectively, when searched through the TESS website. Overexpression of cMyb increased the BC200 RNA transcription and the cMyb siRNA treatment reduced the transcription, suggesting that the -17 to -12 region might be the binding site for cMyb. The BC200 RNA transcription was severely repressed by treatment with TBP siRNA, indicating that TBP is essential for the transcription. Three putative TBP binding sites were observed within the -100 upstream sequence, but only the mutation at the -28 to -22 region severely affected BC200 RNA transcrip-tion. Therefore, it is likely that the -28 to -22 region is the TBP binding site. However, overexpression of c-ETS-2 slightly reduced the BC200 RNA and the c-ETS-2 siRNA treatment increased the transcription, sug-gesting that c-ETS-2 would not be responsible for transcription activation by directly binding to the upstream sequence. Instead, knockdown of MAF with siRNA suppressed BC200 RNA transcription, suggesting that MAF might bind to the -64 to -59 region as a transcription activator. A biotin-streptavidin pool-down experiment was performed to identify proteins binding to the up-stream -118 sequence. Proteins binding to the biotin-labeled DNA fragment were analyzed by MALDI-TOF-MS, and vimentin and hnRNP K were identified as binding proteins. hnRNP K gene silencing increased BC200 transcription, but decreased cellular levels of BC200 RNA. EMSA results showed that hnRNP K could bind to the upstream sequence, but the DNase I foopring failed to identify any specific binding sites on that sequence. This result indicates that hnRNP K might act as a transcription repressor directly or indirectly as well as a metabolic stabilizer for BC200 RNA. Since the treatment with hnRNA K siRNA resulted in the re-duction of endogenous BC200 RNA despite of its transcriptional suppression, metabolic stabilization of BC200 RNA would be the key factor. Knockdown of vimentin with siRNA led to induction of BC200 tran-scription. Therefore, vimentin might play a role as a transcriptional suppressor for BC200 RNA. The effects of vimentin were independent of the upstream sequence, suggesting that vimentin would affect BC200 RNA transcription without binding to the upstream sequence. Effects of proto-oncogenes c-myc and YY1 or tumor suppressor p53 on BC200 RNA biosynthesis were also examined using the respective siRNAs. BC200 RNA transcription was decreased by c-myc and p53 siRNAs, but increased by YY1 siRNA, indicating that tumor-related genes could affect BC200 RNA transcription. Taken all together, this study may provide mechanistic links between aberrant BC200 expression and specific signaling pathways related to tumorigenesis.