Figure 5.

Differential regulation of mRNAs by MSI1/AGO2 complex under hypoxia. (A) Endogenous MSI1 or AGO2 was immunoprecipitated in MSI1 or AGO2 knockdown 05MG cell with anti-MSI1 or anti-AGO2 antibody. Western blot of the immunoprecipitation (IP) confirmed the MSI1/AGO2 interaction in hypoxia-treated parental cells but not in MSI1 or AGO2 knockdown cells (top). Total RNAs isolated from IP were subjected to NF2, TP53, CCND1, and HELLS mRNA quantitation by using qPCR with specific primer. Quantification of mRNA expression levels experiments by normalization with IgG control. Data represent the mean ± S.D. of three independent experiments performed in triplicate. * P<0.05 vs IgG signal. (B) Nuclear and cytosolic fractions of 05MG/MSI1-wt, 05MG/MSI1-NES-mut, and 05MG/MSI1-NLS-mut cells were subjected to the immunoprecipitation with Flag antibodies to pull down the complexes interacting with Flag-tagged MSI1. Left, immunoprecipitates were subjected to Western blot to assess the binding between AGO2 and full-length or mutated MSI1. Right, total RNAs isolated from the immunoprecipitated complexes were analyzed by qRT-PCR for NF2, TP53, CCND1, and HELLS mRNA levels. Fold change in mRNA levels was normalized to IgG-precipitated controls. Data represent the mean ± S.D. of three independent experiments performed in triplicate. * P<0.05 vs IgG signal. (C) Modified-RIP analysis of the binding regions of MSI1 and AOG2 on the target mRNAs. RIP were performed with anti-MSI1 or anti-AGO2 followed by RNA fragmentation and qPCR of NF2, TP53, CCND1, and HELLS coding sequence (CDS) and 3´ UTR. The schematic illustration showed the relative locations of each pair of qPCR primers for CDS and 3'UTR regions. MSI1 or AGO2 palindromic-binding sequence exists within the peak. Quantification of fold changes of the signals were normalized to IgG-precipitated controls. This experiments were done in three distinct biological replicates. (D) A schematic illustrating the fate of mRNA determined by the MSI1-AGO2 complex. MSI1-AGO2 regulates RNA stability of specific RNAs to sustain tumor growth under stress in two ways: 1) MSI1-AGO2 facilitates tumor suppressor gene mRNA decay to prevent stress-induced cell death (likely through the conventional UTR binding followed by post-transcriptional repression) and 2) MSI1-AGO2 stabilizes and protects mRNA of cell cycle genes to promote prompt translation upon stress removal (likely through the CDS binding and subsequent aggregation in stress granules).