Oncogenic K-Ras suppresses global miRNA function.

K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets.

The impact of the complexing agent on the sensitivity of collision-induced dissociation spectra to fatty acid position for a set of XYZ-type triglycerides.

RATIONALE: The development of an automated platform for the positional analysis of triglycerides (TAGs) based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) continues to be pursued. This work evaluates the positional sensitivities of the collision-induced dissociation (CID) spectra of a representative set of XYZ triglycerides using sodium, lithium, and ammonium salts as complexing agents. METHODS: A set of triglycerides were synthesized and analyzed via ESI-MS/MS using an ion trap mass spectrometer. Using three different complexing agents, the product ion spectra of the corresponding precursor ions for twelve XYZ TAGs were collected, where X, Y, and Z represent C16:0 , C18:1(c-9) , C18:2(cc-9,12) , and C20:4(cccc-5,8,11,14) fatty acid chains. These data were then used to prepare ternary plots for four positional isomer systems to evaluate the positional sensitivity differences among the three different complexing agents. RESULTS: The positional sensitivities for each of the four positional isomer systems were robust for the sodium and lithium adducts. The CID data for the sodium and lithium TAGs demonstrated an unfavorable loss of the fatty acid in the center position and showed a higher sensitivity to fatty acid position, when compared with the CID data for ammonium adducts, especially for the arachidonic acid containing triglycerides. CONCLUSIONS: The data shows that that the relative abundances of the DAG product ions for the XYZ-type TAGs when using sodium and lithium complexing agent adducts are sensitive to fatty acid position and are consistent for the diverse array of TAGs studied in this work. This suggests that using sodium or lithium as the complexing agent may be advantageous for the development of an automated platform for the positional analysis of complex TAG mixtures based on ESI-MS/MS.