Optimized proximity ligation assay (PLA) for detection of RNA-protein complex interactions in cell lines

George, Jasmine; Mittal, Sonam; Kadamberi, Ishaque P.; Pradeep, Sunila; Chaluvally‐Raghavan, Pradeep

doi:10.1016/j.xpro.2022.101340

Cited by 9 publications

(3 citation statements)

References 10 publications

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“…After 2 days, cells were transfected with Myc-B1B2C and His-PDE5 or co-transfected with both Myc-B1B2C and His-PDE5 clones for 24 h following a published PLA protocol. 22 The cells were washed 3 times with PBS, fixed in 4% PFA in PBS for 10 min, permeabilized by ice-cold methanol for 20 min, washed 3 times with PBS, blocked with 3% BSA in PBS, washed again with PBS, and incubated with appropriate primary antibodies for 16 h at 4°C. Cells were washed with 0.1% PBST and incubated with the appropriate amount of anti-mouse MINUS and anti-rabbit PLUS probes supplied with Duolink in situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma–Aldrich, USA), according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination

et al. 2023

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We identified Rho Related BTB Domain Containing 1 (RhoBTB1) as a key regulator of phosphodiesterase 5 (PDE5) activity, and through PDE5, a regulator of vascular tone. We identified the binding interface for PDE5 on RhoBTB1 by truncating full-length RhoBTB1 into its component domains. Co-immunoprecipitation analyses revealed that the C-terminal half of RhoBTB1 containing its two BTB domains and the C-terminal domain (B1B2C) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via the Cullin-3 (CUL3). The C-terminal domain was essential in recruiting PDE5 as constructs lacking this region could not participate in PDE5 binding or proteasomal degradation. We also identified Pro353 and Ser363 as key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of either of these residues exhibited impaired CUL3 binding and PDE5 degradation, although the binding to PDE5 was preserved. Finally, we employed ascorbate peroxidase 2 (APEX2) proximity labeling using a B1B2C-APEX2 fusion protein as bait to capture unknown RhoBTB1 binding partners. Among several B1B2C-binding proteins identified and validated, we focused on SET Domain Containing 2 (SETD2). SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to pharmacological inhibition of the proteasome or Cullin complex, CUL3 deletion, and RhoBTB1-inhibition with siRNA. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies will determine if SETD2 plays a role in cardiovascular function.

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Section: Methodsmentioning

confidence: 99%

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination

et al. 2023

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“…It can be adapted to detect RNA: protein interactions if RNA can be detected with a dye, for example, by using an RNA probe labeled with digoxigenin to enable the detection of endogenous RNAs using antibodies. However, to date, very few studies have benefited from this method to explore RNA: protein interactions ( Roussis et al, 2016 ; Zhang et al, 2016 ; George et al, 2022 ). First, the cost of secondary antibodies and reagents to achieve amplification is expensive, and it is therefore unlikely that high-throughput screens could be used under these conditions.…”

Section: Cellular Assaysmentioning

confidence: 99%

Using the structural diversity of RNA: protein interfaces to selectively target RNA with small molecules in cells: methods and perspectives

Li,

Bouhss,

Clément

et al. 2023

Front. Mol. Biosci.

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In recent years, RNA has gained traction both as a therapeutic molecule and as a therapeutic target in several human pathologies. In this review, we consider the approach of targeting RNA using small molecules for both research and therapeutic purposes. Given the primary challenge presented by the low structural diversity of RNA, we discuss the potential for targeting RNA: protein interactions to enhance the structural and sequence specificity of drug candidates. We review available tools and inherent challenges in this approach, ranging from adapted bioinformatics tools to in vitro and cellular high-throughput screening and functional analysis. We further consider two critical steps in targeting RNA/protein interactions: first, the integration of in silico and structural analyses to improve the efficacy of molecules by identifying scaffolds with high affinity, and second, increasing the likelihood of identifying on-target compounds in cells through a combination of high-throughput approaches and functional assays. We anticipate that the development of a new class of molecules targeting RNA: protein interactions to prevent physio-pathological mechanisms could significantly expand the arsenal of effective therapeutic compounds.

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“…This PDF file includes: Materials and Methods Figs. S1 to S9 Tables S1 to S10 references (76)(77)(78) Other Supplementary Material for this manuscript includes the following: Data file S1 MDar reproducibility checklist…”

Section: Supplementary Materialsmentioning

confidence: 99%

Wild-type IDH1 maintains NSCLC stemness and chemoresistance through activation of the serine biosynthetic pathway

Zhang,

Yu,

Yang

et al. 2023

Sci. Transl. Med.

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Tumor-initiating cells (TICs) reprogram their metabolic features to meet their bioenergetic, biosynthetic, and redox demands. Our previous study established a role for wild-type isocitrate dehydrogenase 1 (IDH1 WT ) as a potential diagnostic and prognostic biomarker for non–small cell lung cancer (NSCLC), but how IDH1 WT modulates NSCLC progression remains elusive. Here, we report that IDH1 WT activates serine biosynthesis by enhancing the expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1), the first and second enzymes of de novo serine synthetic pathway. Augmented serine synthesis leads to GSH/ROS imbalance and supports pyrimidine biosynthesis, maintaining tumor initiation capacity and enhancing gemcitabine chemoresistance. Mechanistically, we identify that IDH1 WT interacts with and stabilizes PHGDH and fragile X–related protein-1 (FXR1) by impeding their association with the E3 ubiquitin ligase parkin by coimmunoprecipitation assay and proximity ligation assay. Subsequently, stabilized FXR1 supports PSAT1 mRNA stability and translation, as determined by actinomycin D chase experiment and in vitro translation assay. Disrupting IDH1 WT -PHGDH and IDH1 WT -FXR1 interactions synergistically reduces NSCLC stemness and sensitizes NSCLC cells to gemcitabine and serine/glycine–depleted diet therapy in lung cancer xenograft models. Collectively, our findings offer insights into the role of IDH1 WT in serine metabolism, highlighting IDH1 WT as a potential therapeutic target for eradicating TICs and overcoming gemcitabine chemoresistance in NSCLC.

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Optimized proximity ligation assay (PLA) for detection of RNA-protein complex interactions in cell lines

Cited by 9 publications

References 10 publications

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination

Using the structural diversity of RNA: protein interfaces to selectively target RNA with small molecules in cells: methods and perspectives

Wild-type IDH1 maintains NSCLC stemness and chemoresistance through activation of the serine biosynthetic pathway

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