In vivo RNA Affinity Purification of wildtype and nonstop mutant VHL tumor suppressor mRNA

ABSTRACT: Nonstop extension or stop-loss mutations in the von Hippel Lindau (VHL) tumor suppressor gene are recurrently found in kidney cancer patients as observed from the NonStopDB database. We observed that presence of this nonstop extension mutation caused loss of the protein via the ubiquitin-proteasome pathway. Stabilization of the nonstop extended VHL with proteasome inhibitor Bortezomib revealed that the mutant protein was preferentially translated from an upstream alternative start site compared to the wild-type. The objective of this study was to identify proteins bound to the VHL mRNA that could be responsible for the differential start site selection. For this, we employed the in vivo RNA-affinity purification method to pull down wild-type and nonstop mutant VHL mRNAs along with their bound proteins after cross-linking using UV irradiation and pull-down with raPOOLs, which are a pool of biotinylated DNA oligonucleotides reverse complementary to the VHL mRNA. The RNA-protein complexes were then pulled down by streptavidin beads, the bound proteins were isolated and subjected to mass-spectrometry based identification.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Sven Diederichs  

LAB HEAD: Sven Diederichs

PROVIDER: PXD053175 | Pride | 2025-05-07

REPOSITORIES: Pride