Long noncoding RNA HOTAIR interacts with Y-Box Protein-1 (YBX1) to regulate cell proliferation

Abstract

HOTAIR is a long noncoding RNA (lncRNA) which serves as an important factor regulating diverse processes linked with cancer development. Here, we used comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) to explore the HOTAIR-protein interactome. We were able to identify 348 proteins interacting with HOTAIR, allowing us to establish a heavily interconnected HOTAIR-protein interaction network. We further developed a novel near-infrared fluorescent protein (iRFP)-trimolecular fluorescence complementation (TriFC) system to assess the interaction between HOTAIR and its interacting proteins. Then, we determined that HOTAIR specifically binds to YBX1, promotes YBX1 nuclear translocation, and stimulates the PI3K/Akt and ERK/RSK signaling pathways. We further demonstrated that HOTAIR exerts its effects on cell proliferation, at least in part, through the regulation of two YBX1 downstream targets phosphoenolpyruvate carboxykinase 2 (PCK2) and platelet derived growth factor receptor β. Our findings revealed a novel mechanism, whereby an lncRNA is able to regulate cell proliferation via altering intracellular protein localization. Moreover, the imaging tools developed herein have excellent potential for future in vivo imaging of lncRNA–protein interaction.

Systematic Survey of the Regulatory Networks of the Long Noncoding RNA BANCR in Cervical Cancer Cells

“…Several studies have reported that lncRNAs apply diverse molecular mechanisms to regulate a wide range of biological functions, including linking to mRNA transcription of target genes or interacting with some RNA-binding proteins directly. − Here, we found that BANCR regulates the expression of CREB1 at the protein level but not at the mRNA level. Moreover, our RNA pull-down experiment revealed that BANCR may not directly interact with CREB1.…”

Orthogonal Proteomics Methods to Unravel the HOTAIR Interactome

“…During RNA-BioID, however, we overexpressed HOTAIR to much higher levels, suggesting that the expression level of HOTAIR might be crucial to detect PRC2 and LSD1 complexes. Li et al 27 performed ChIRP-MS to identify HOTAIR-interacting proteins in whole cell lysates of HeLa cells, which express even lower amount of endogenous HOTAIR compared to MCF7. Similarly, they did not identify PRC2 or LSD1 complex subunits and attributed this to highly abundant cytoplasmic proteins masking the LC-MS/MS signal of these known interactors.…”

The smallest near-infrared fluorescence complementation system for imaging protein–protein and RNA–protein interactions

“… 17 The pmiRC108-L-MCP plasmid was produced by replacing the iRFP fragment iRC124 m in piRC124 m -MCP with miRC108 using restriction enzymes Nhe I and Hind III. 29 The pNS1-2*L-miRN107/pNS1-3*L-miRN107 plasmids and pmiRC108-2*L-MCP/pmiRC108-3*L-MCP plasmids with different linker lengths within the fused proteins were constructed in a similar manner to the pNS1-L-miRN107 and pmiRC108-L-MCP plasmids.…”

“…The protein expression plasmids were generated based on mIrisFP and iRFP TriFC systems. 17,29 The fragments of miRN107 and miRC108 were produced using the pUC57-miRFP670nano plasmid (Genewiz, Suzhou) as the template. The pNS1-L-miRN107 plasmid was then generated by replacing the mIrisFP fragment mIN165 in pNS1-mIN165 with miRN107 using restriction enzymes KpnI and XbaI.…”

“…5,[7][8][9][10] In particular, the introduction of red, far-red and near-infrared uorescent emission proteins (i.e., green uorescence protein (GFP)-like and phytochromes) has expanded the spectrum of signal modules acting as molecular sensors and facilitated in vivo applications [6][7][8][11][12][13][14] because the mammalian body has low tissue autouorescence, low light scattering and minimal absorbance of melanin, water and hemoglobin in the nearinfrared spectral region of $600-1200 nm, which is termed the "optical window" of mammalian tissue. 15 Currently, BiFC systems are primarily developed from GFPlike proteins or near-infrared phytochromes, which are large macromolecules (27)(28)(29)(30)(31)(32)(33)(34)(35)). 6,13,14 The fusion of large proteins has raised the question of whether the function of each protein is affected by its fusion partner.…”