Characterization of interaction between the long non-coding RNA, Brain Cytoplasmic RNA 1 (BCYRN1, BC200) and Signal Recognition Particle 9/14 (SRP9/14)
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Date
2020
Authors
Choi, Taegi
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Abstract
Brain cytoplasmic 200 (BC200) is a primate-specific neural long non-coding RNA (lncRNA) with a length of 200 nucleotides. BC200 RNA is comprised of three different segments, with the first 120 nucleotides homologous to left monomeric Alu-J element, followed by a poly adenosine rich region and lastly a unique 40 nucleotide long 3’ cytosine rich region. BC200 has been shown to be overexpressed in tumor cells and plays a role in inhibition of cellular apoptotic pathways. The function of BC200 was hypothesized to be mediating gene regulation in cells, however, no direct function has been determined. To better understand BC200’s function, my laboratory had previously validated BC200 Ribonucleoprotein (RNP) complexes by proteomic analysis using recombinant BC200 transfection. The validation of binding partners from the proteomic analysis had revealed that the heterodimeric signal recognition particle (SRP) 9 and 14 are highly specific towards a specific region of BC200 (Alu element). To further understand the SRP9/14 interaction with BC200 and to potentially uncover a novel BC200 function, in vitro studies of SRP9/14 was sought. The validation of SRP9/14 - BC200 interactions was conducted via optimization of expression and purification of SRP9/14 in both bacterial and human cells. BC200 was transcribed and purified using theT7 RNA polymerase system in vitro to pursue biophysical studies of SRP9/14-BC200 interactions. Lastly, knock-down studies of SRP9/14 were conducted with respect to BC200 in MCF-7 cells and confirmed that the protein levels of SRP9 and SRP14 were co-dependent which upon the reduction of one, the other was also reduced. RNA immunoprecipitation was then conducted on SRP9/14 knockdown in MCF-7 cells and demonstrated a reduction in interaction between CSDE1 and BC200. Changes in the cellular translation rate was also measured using the puromycin assay upon SRP9 knock-down, which had demonstrated a reduction in translation rate mirroring the effect of BC200 knock-down in similar conditions. In summary, the characterization of BC200-SRP9/14 was attempted at varying directions to better investigate functional aspects of BC200 and its RNP formations.
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Keywords
LncRNA, BC200, RNP, SRP9/14
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APA