Many non-coding RNAs (ncRNAs) serve as regulatory molecules in various physiological pathways, including gene expression in mammalian cells. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. It is thus important to understand transcriptional regulation in ncRNA genes but is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA polymerase II (Pol II) promoter-proximal pausing and in a tissue-specific manner. In this study, human ncRNA genes that are expressed in the early G 1 phase, termed immediate early ncRNA genes, were monitored to assess the function of positive transcription elongation factor b (P-TEFb), a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is induced in the G 0 –G 1 transition and regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb-dependent transcription of serum responsive ncRNA genes was observed: Pol II carboxyl-terminal domain phosphorylated at serine 2 (S2) was largely increased in the transcription start site (TSS, -300 to +300) whereas overall, it was decreased in the gene body (GB, > +350) upon chemical inhibition of P-TEFb. In addition, the three representative, immediate early ncRNAs, whose expression is dependent on P-TEFb, metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ), nuclear enriched abundant transcript 1 ( NEAT1 ), and X-inactive specific transcript ( XIST ), were further analyzed for determining P-TEFb association. Taken together, our data suggest that transcriptional activation of many human ncRNAs utilizes the pausing and releasing of Pol II, and that the regulatory mechanism of transcriptional elongation in these genes requires the function of P-TEFb. Furthermore, we propose that ncRNA and mRNA transcription are regulated by similar mechanisms while P-TEFb inhibition unexpectedly increases S2 Pol II phosphorylation in the TSSs in many ncRNA genes. One Sentence Summary: P-TEFb regulates Pol II phosphorylation for transcriptional activation in many stimulus-inducible ncRNA genes.

Methyl benzoate (MB) is a small, hydrophobic organic compound that is isolated from the freshwater fern, Salvinia molesta. Because of its pleasant odor, it has been used as a fragrance and flavor enhancer. In addition, it is used to attract orchid bees for pollination in the farm and has been tested for its potential to be developed as a green pesticide targeting a diverse group of insects. In spite of its wide applications, the safety of MB to humans remains poorly understood. In this study, we tested the cytotoxicity of MB against cultured human cells, including kidney, colon, and neuronal cells. Furthermore, other natural and synthetic benzoic acids such as ethyl benzoate (EB) and vinyl benzoate (VB) were compared with MB for their similarity and broad commercial and industrial applications. We found that MB and VB have the least and most overall toxicity to the tested human cells, respectively. In addition, the expression of some genes involved in cell cycle, protein quality control, and neurotransmission such as cyclin D1, HSP70, and ACHE genes was differentially expressed in the presence of these chemicals, most noticeably in treatment of VB. Our study provided the LC 50 values of these benzoic acids for human cells in vitro and suggested their mild toxicity that should be considered in the industrial and agricultural applications to be within safe limits.

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