Functional Analysis of RNA Motifs Essential for BC200 RNA-mediated Translational Regulation

Abstract

Brain cytoplasmic 200 RNA (BC200 RNA) is proposed to act as a local translational modulator by inhibiting translation after being targeted to neuronal dendrites. However, the mechanism by which BC200 RNA inhibits translation is not fully understood. Although a detailed functional analysis of RNA motifs is essential for understanding the BC200 RNA-mediated translation-inhibition mechanism, there is little relevant research on the subject. Here, we performed a systematic domain-dissection analysis of BC200 RNA to identify functional RNA motifs responsible for its translationalinhibition activity. Various RNA variants were assayed for their ability to inhibit translation of luciferase mRNA in vitro. We found that the 111-200-nucleotide region consisting of part of the Alu domain as well as the A/C-rich domain (consisting of both the A-rich and C-rich domains) is most effective for translation inhibition. Surprisingly, we also found that individual A-rich, A/C-rich, and Alu domains can enhance translation but at different levels for each domain, and that these enhancing effects manifest as cap-dependent translation. [BMB Reports 2020; 53(2): 94-99] BMB Rep. 2020; 53(2): 94-99 www.bmbreports.org

The noncoding RNA BC200 associates with polysomes to positively regulate mRNA translation in tumor cells

“…We have recently reported the interaction of BC200 with a number of protein binding partners that supports the proposed function of BC200 as a regulator of mRNA translation ( 34 ). As previous studies looked exclusively at translational changes in response to overexpression of BC200 ( 28 , 29 , 30 , 31 , 32 , 33 ), we sought to assess the impact of BC200 knockdown on mRNA translation rates. Knockdown of BC200 in MCF-7 cells with an LNA GapmeR resulted in a significant reduction in translation rates within 12 h as measured by puromycin incorporation ( Fig.…”

“…In tumor cell line models, BC200 is critical for cell viability as well as to promote cell migration and invasion ( 7 , 24 , 25 , 26 , 27 ). In terms of specific function, overexpression studies of both BC200 and BC1 have suggested a role for both RNAs in negative regulation of translation in both in-cell as well as in vitro translation assays ( 28 , 29 , 30 , 31 , 32 , 33 ). Supporting these reports, we have previously described the interaction of BC200 with a number of proteins that implicate potential roles in mRNA stability, translation, and splicing (CSDE1, DHX36, PABPC1, PABPN1, HNRNPK, SRP9/14, SYNCRIP) ( 17 , 34 ).…”

“…MCF-7 cells were selected for further study as they exhibit elevated BC200 expression, rely on BC200 for cell viability, and have been used to assess the protein components of the BC200 RNP ( 7 , 34 ). BC200 has previously been reported as a negative regulator of translation; however, these studies involved either transfection of in vitro transcribed RNA ( 30 , 31 ) or the use of non-primate-based in vitro translation assays ( 28 , 29 , 30 , 33 , 44 ). Although we were able to replicate these results in-cell by transfection of in vitro transcribed BC200, a scrambled control RNA produced a more robust effect.…”