UV-crosslinking mapping of LARP6 interaction sites with RNA

ABSTRACT: To map the regions of LARP6 that interacts directly with RNA, we developed a mass spectrometry-based approach termed IP-OOPS (Immunoprecipitation coupled with Orthogonal Organic Phase Separation. This approach combines the isolation of UV-C crosslinked RNA–protein adducts via Orthogonal Organic Phase Separation, with the ability to map protein regions that are attached to RNA by sequential Arg-C or Lys-C digestion, followed by trypsin digestion after purification of RNA-bound fragments, as pioneered in the RBDmap method (Castello et al., 2016). We used GFP-trap beads were used to purify LARP6-RNA complexes from UV-crosslinked MDA-MB231 cells that stably expressed GFP-LARP6 (Dermit et al., 2020). The RNA-crosslinked LARP6 was then isolated from the immunoprecipitated LARP6 fraction, using an initial OOPS, before partial digestion with Arg-C or Lys-C, which generated smaller segments, with or without RNA adducts. The RNA-bound segments were further purified from the unbound segments via a second OOPS, followed by trypsin digestion, releasing MS-detectable peptides that were adjacent to a crosslinked region. The crosslinked peptides themselves are undetectable in this approach, due to the addition of the unknown RNA mass, but released peptides after the second phase separation can be readily identified, which map to regions adjacent to the crosslink sites. In our study, Crosslink-adjacent peptides were only detected from the initial Arg-C digests.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Faraz Mardakheh  

LAB HEAD: Faraz Mardakheh

PROVIDER: PXD064029 | Pride | 2026-01-22

REPOSITORIES: Pride