Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR

Abstract

BC200 is a long non-coding RNA primarily expressed in brain but aberrantly expressed in various cancers. To gain a further understanding of the function of BC200, we performed proteomic analyses of the BC200 ribonucleoprotein (RNP) by transfection of 3′ DIG-labelled BC200. Protein binding partners of the functionally related murine RNA BC1 as well as a scrambled BC200 RNA were also assessed in both human and mouse cell lines. Stringent validation of proteins identified by mass spectrometry confirmed 14 of 84 protein binding partners and excluded eight proteins that did not appreciably bind BC200 in reverse experiments. Gene ontology analyses revealed general roles in RNA metabolic processes, RNA processing and splicing. Protein/RNA interaction sites were mapped with a series of RNA truncations revealing three distinct modes of interaction involving either the 5′ Alu-domain, 3′ A-rich or 3′ C-rich regions. Due to their high enrichment values in reverse experiments, CSDE1 and STRAP were further analyzed demonstrating a direct interaction between CSDE1 and BC200 and indirect binding of STRAP to BC200 via heterodimerization with CSDE1. Knock-down studies identified a reciprocal regulatory relationship between CSDE1 and BC200 and immunofluorescence analysis of BC200 knock-down cells demonstrated a dramatic reorganization of CSDE1 into distinct nuclear foci.

The role of CSDE1 in translational reprogramming and human diseases

“…Immunoprecipitation assays showed that knocking down Unrip had a negligible impact on lncBC200 binding to CSDE1. However, knocking down CSDE1 in MCF-7 cells reduced Unrip binding on lncBC200 by more than 80% [64]. These results suggest the existence of a sequential connection of "Unrip-CSDE1-lncBC200".…”

“…LncBC200 is a 200-nucleotide ncRNA that is normally highly expressed in the brain, but it is aberrantly expressed in various cancers [63]. In MCF-7 cells, CSDE1 could maintain the stability of lncBC200 by binding to the 3' A-rich region, while knocking down CSDE1 reduced the half-life of lncBC200 by 40% and decreased its expression [64].…”

“…In this mechanism, CSDE1 facilitates the binding of other regulators to RNA by adjusting the IRES structure [39,42]. In a few cases, CSDE1 connects indirect regulators with their target RNAs based on the sequential connection mode [64]. For all modes, CSDE1 binds RNAs directly and cooperates with other regulators in direct or indirect ways.…”

Current approaches for RNA-labelling to identify RNA-binding proteins

“…The DIG-labelled RNA probe can then be incubated with cellular extract, and then placed to incubate onto magnetic beads. These magnetic beads are conjugated with a chemiluminescent anti-DIG antibody that is used to immunoprecipitate the potential RBPs associated with the DIG-labelled RNA (Booy et al 2018). Herranz and Pallás (2004) demonstrated, using DIG-labelled riboprobes, that the movement protein interacts with the Prunis necoritic ringsport viral ssRNA.…”

An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21

“…Native RNA immunoprecipitations were performed on one 150 mm dish of HEK293T cells as previously described 45 degrees Celsius in TBST+ 5% milk, GAPDH and tubulin primary antibodies were incubated for 1 hour at room temperature in TBST+5% milk. Three sequential 5-minute washes with 5% TBS-T were followed by 1-hour incubation with secondary antibodies (1:10000) in TBST+5% milk and then 4 more 10-minute washes with TBS-T.…”