An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene

Azman, Muhammad S.; Dodel, Martin; Capraro, Federica; Faraway, Rupert; Dermit, Maria; Fan, Wanling; Ule, Jernej; Mardakheh, Faraz K.

doi:10.1101/2021.12.16.472890

Cited by 3 publications

(10 citation statements)

References 88 publications

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“…Based on this pattern of interaction, we hypothesized that, rather than being involved in a specific step of rRNA processing, members of cluster 9 may have a role in regulating rRNA synthesis. To assess this hypothesis, we used RNAi to deplete ten proteins in the cluster 9 that were not previously implicated in ribosome biogenesis, and assessed the impact on rRNA synthesis by nascent RNA imaging 61 . A significant reduction in nascent rRNA levels was detected upon depletion of 9 out of 10 proteins (Extended Data Fig.…”

Section: Trex Reveals Region-specific and Multiregional Rrna Bindersmentioning

confidence: 99%

“…Acetone-precipitated proteins from TREX were subjected to in-solution digestion by trypsin as described previously 61 . Briefly, proteins were recovered in 200 µl of 2 M urea, 50 mM ammonium bicarbonate and reduced by adding dithiothreitol to a final concentration of 10 mM.…”

Section: Ms Sample Preparation and Data Acquisitionmentioning

confidence: 99%

“…Fractions were dried with vacuum centrifugation before recovery in A* buffer for LC-MS/MS analysis. LC-MS/MS analysis was performed as described before 61 , using a Q Exactive-plus Orbitrap mass spectrometer coupled with a nanoflow ultimate 3000 RSL nano HPLC platform (ThermoFisher). The instrument was operated using the Thermo Xcalibur v.4.5 SP1 software.…”

Section: Ms Sample Preparation and Data Acquisitionmentioning

confidence: 99%

“…CLIP was performed as described previously 61 , with some modifications. Briefly, HCT116 cells were irradiated on ice with 150 mJ cm 2 UV light (254 nm) in PBS, using a Hoefer Scientific UV crosslinker.…”

Section: Crosslinking and Immunoprecipitationmentioning

confidence: 99%

“…All samples were diluted 1:1 with RNAse-free water and 2 µl were used as input for quantitative real-time RT-qPCR analysis. In parallel, the aliquots of input and immunoprecipitate that were taken for protein analyses were resuspended in lithium dodecyl sulfate sample buffer and subjected to western blotting with the indicated antibodies, as described previously 61 .…”

Section: Crosslinking and Immunoprecipitationmentioning

confidence: 99%

See 4 more Smart Citations

TREX reveals proteins that bind to specific RNA regions in living cells

Dodel,

Guiducci,

Dermit

et al. 2024

Nat Methods

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Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells and under endogenous settings. Here we introduce TREX (targeted RNase H-mediated extraction of crosslinked RBPs)—a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. With its applicability to different cell types, TREX is an RNA-centric tool for unbiased positional mapping of endogenous RNA–protein interactions in living cells.

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Section: Trex Reveals Region-specific and Multiregional Rrna Bindersmentioning

confidence: 99%

Section: Ms Sample Preparation and Data Acquisitionmentioning

confidence: 99%

Section: Ms Sample Preparation and Data Acquisitionmentioning

confidence: 99%

Section: Crosslinking and Immunoprecipitationmentioning

confidence: 99%

Section: Crosslinking and Immunoprecipitationmentioning

confidence: 99%

See 3 more Smart Citations

TREX reveals proteins that bind to specific RNA regions in living cells

Dodel,

Guiducci,

Dermit

et al. 2024

Nat Methods

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CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature ofKRASandNRASmutant cancers

Basu

Luke

Karim

et al. 2022

Preprint

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Oncogenic RAS induces perinuclear translocation of the oncogenic kinases ERK1/2 and CK2 and the MAPK scaffold KSR1, forming endosomal signaling hubs termed perinuclear signaling centers (PSCs). PSCs are found in nearly all cancer cell lines and tumor tissues, suggesting that compartmentalization of oncogenic kinases drives tumorigenesis. We show here that the endosomal adaptor, TOLLIP, tethers RAB11A+ signaling endosomes containing CK2 and KSR1, but not ERK, to the perinuclear ER. TOLLIP binds to the KSR1 CA5 pseudo-kinase domain through a conserved region predicted to form a β-hairpin, thus anchoring PSCs to perinuclear endosomes. TOLLIP is perinuclear in cancer cells and KRasG12D-driven mouse tumors but is pan-cytoplasmic in non-transformed cells, correlating with the presence of PSCs. TOLLIP is required for proliferation/survival of KRAS mutant tumor cells but not HRAS, NRAS and BRAF cancers or non-transformed cells. KRasG12D-induced lung lesions in TollipKO/KO mice showed reduced numbers of carcinomatous lesions, implicating TOLLIP in progression to malignant cancer. Phosphoproteomics studies revealed that perinuclear CK2 phosphorylates specific substrates, particularly proteins involved in ribosome biogenesis and translation such as RIOK1 and eIF4B. In summary, our findings identify TOLLIP as a key signaling adaptor in KRAS tumors whose inhibition is a potential vulnerability of these cancers.

show abstract

TREX reveals proteins that bind to specific RNA regions in living cells

Dodel

Guiducci

Dermit

et al. 2023

Preprint

View full text Add to dashboard Cite

Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells under endogenous settings. Here, we introduce TREX (Targeted RNase H-mediated extraction of crosslinked RBPs), a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD lncRNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and novel interactions that regulate ribosome biogenesis. With its applicability to any RNA in any cell-type, TREX is the first RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells.

show abstract

An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene

Cited by 3 publications

References 88 publications

TREX reveals proteins that bind to specific RNA regions in living cells

TREX reveals proteins that bind to specific RNA regions in living cells

CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature ofKRASandNRASmutant cancers

TREX reveals proteins that bind to specific RNA regions in living cells

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