Development And Optimization Of A High Content Screening Assay To Discover Novel Small Molecules Regulating Paraspeckles Function

Paraspeckles, a relatively new class of subnuclear bodies found in the interchromatin space of mammalian cells, are enriched with noncoding KNA-NEAT1and RNA binding proteins. And the subnuclear bodies can precipitate the nuclear retention of RNAs containing double-stranded RNA regions that have been subject to adenosine-to-inosine (A-to-I) editing in their3'untranslated regions (3'UTRs). As a result, these RNAs are prevented from being translated into proteins to play a biological function. So, paraspeckles could block the signal conversion of gene transcription to functional protein translation through nuclear retention of RNAs, but do not affect these processes. At the same time, paraspeckles could control target gene expression accurately and rapidly by regulating mRNA nuclear retention or nuclear export which is different from gene transcription or protein translation. Under physiologic stress, CTN-RNA is posttranscriptionally cleaved in its3'UTRs localizing to paraspeckles, and the released mRNA, similar to mCAT2mRNA, is transported to the cytoplasm and translated into mCAT2protein. This mechanism provides a rapid response for the production of mCAT2protein to modulate the cellular uptake of L-arginine for the NO pathway.Furthermore, human embryonic stem cells (hESCs) are the only cells that have little or no detectable NEAT1RNA and do not show paraspeckles. However, when the hESCs are induced to differentiate, NEAT1is expressed and paraspeckles appear. Importantly, the appearance of paraspeckles is linked to an increase in the efficiency of nuclear retention of the A-to-I edited mRNAs. Meanwhile, VINC-1RNA (subsequently found to correspond to NEAT1) increase in the central nervous system of mice infected with Japanese encephalitis or rabies viruses, and these viruses may also trigger an increase in paraspeckles size and number. Thus, paraspeckles may have an important role in controlling gene expression during many cellular processes including stress responses, differentiation and viral infection. At present, the function and mechanism of paraspeckles in controlling gene expression remains to be determined. Therefore, this thesis took paraspeckles prevalence increase or reduction as a phenotypic alternation marker of paraspeckles and established a high content screening assay to identify novel small molecules that regulate paraspeckles function. And these novel small molecules could be used to elucidate the nature of the paraspeckles function.Here we established a cell line of HEK293(human embryonic kidney293) stably expressing an EYFP (enhanced yellow fluorescent protein)-fusion protein, EYFP-PSPla, that localizes to the paraspeckles. Then, using Q-PCR,western blotting,fluorescence chromosomal in situ hybridization and immunocytochemistry, we not only certified there was no variability of expression levels of NEAT1,P54NRB and PSF between HEK293(EYFP-PSPla) stable cell line and HEK293cell line, but also found EYFP-PSPla was co-localized with NEAT1,P54NRB or PSF in paraspeckles. So, in this HEK293stable cell line, EYFP-PSPla could effectively localize the position and number of paraspeckles. Furthermore, the stable cell assay was optimized using several high content screening systems. And the number of paraspeckles in each cell was confirmed to be stable and homogeneous. After optimization, the fidelity of the assay was probed with a collection of physiological active nature products library (2560compounds) and biologically active molecules library including1707function-known pharmacologically active molecules, and was shown to be robust and capable of detecting novel paraspeckles-increasing or reducing compounds.135hits, which could markedly increase or reduce paraspeckles expression and with less cytotoxicity, were discovered from the4267compounds. So, this high content screening assay is capable of detecting paraspeckles prevalence in cells, and could provide effectively means to research the composition and function of paraspeckles, making it a significant reported high content phenotypic screen for small changes in nuclear structure.