RNF181 Human

What is RNF181 and what cellular functions does it regulate?

RNF181 is a RING finger-containing protein that functions as an E3 ubiquitin ligase, regulating multiple cellular processes through protein-protein interactions and ubiquitination activities. It contains a characteristic RING finger domain that enables its ubiquitin ligase function. RNF181 has been found to regulate signaling pathways including NF-κB activation in lymphocytes, Hippo/YAP signaling in breast cancer cells, and protein localization in human embryonic stem cells . Methodologically, its function has been confirmed through knockdown experiments, co-immunoprecipitation studies, and ubiquitination assays across various cell types.

How is RNF181 detected in experimental systems?

RNF181 protein can be detected using several complementary methods:

• Western blotting using specific anti-RNF181 antibodies

• Immunoprecipitation followed by mass spectrometry

• Fluorescence tagging (e.g., YPet or FLAG tags) for visualization in live cells

• Bioluminescence resonance energy transfer (BRET) assays for detecting protein-protein interactions in live mammalian cells

For interaction studies, BRET-based interaction cloning (BRIC) has proven valuable for identifying RNF181 binding partners such as CARD11 in lymphocytes. This methodology can detect protein-protein interactions in live mammalian cells in a high-throughput manner using Rluc8 fusion proteins and YPet-tagged potential interactors .

What role does RNF181 play in human embryonic stem cells?

In human embryonic stem cells (hESCs), RNF181 shows specialized interactions distinct from those in differentiated cells. Proteomic analyses have revealed that RNF181 interacts with components of the dynactin complex (specifically DCTN2, ACTR1A, ACTR1B, DCTN4, and DCTN1), which regulates intracellular transport, chromosome alignment, and spindle organization during cell division . Additionally, RNF181 interacts with:

• DZIP3, an E3 enzyme regulating developmental genes through 3D chromatin reorganization

• PSMD3 (a proteasome subunit)

• Subunits of the TRiC/CCT chaperonin complex critical for hESC proliferation and differentiation

Notably, these interaction patterns change during differentiation, with only 17% of RNF181 interactors in hESCs remaining as significant interactors in post-mitotic neuronal cells. This suggests a stem cell-specific function of RNF181 .

How does RNF181 knockdown affect human embryonic stem cell proteome?

RNF181 knockdown in hESCs affects the proteome to a lesser extent than knockdown of other E3 ligases like UBR7 and UBE3A, with 202 proteins showing altered expression . Proteins dysregulated following RNF181 knockdown are primarily involved in mRNA processing-related pathways, which is particularly relevant as transcriptional hyperactivity is considered a hallmark of pluripotent stem cells . mRNA-related proteins deregulated in RNF181 knockdown lines (such as SNRPD1, SRSF5, and HNRNPK) have been shown to be necessary for stemness in pluripotent cells .

Interestingly, only a marginal number of the known RNF181 interacting partners show increased expression upon RNF181 knockdown, suggesting that RNF181 may regulate cellular processes in ways beyond targeting proteins for degradation .

How does RNF181 contribute to breast cancer progression?

RNF181 has been implicated in breast cancer progression through two distinct mechanisms, depending on the breast cancer subtype:

• In ERα-positive breast cancer (70% of breast malignancies):

  • RNF181 stabilizes ERα protein and promotes breast cancer progression

  • RNF181 expression correlates with ERα levels in human breast tumors

  • It associates with ERα and promotes its stability by inducing K63-linked polyubiquitination

  • RNF181 depletion reduces ERα protein levels and target gene expression (e.g., PS2 and GREB1)

• In triple-negative breast cancer (TNBC):

  • RNF181 functions as a positive mediator for Hippo/YAP signaling

  • It interacts with YAP protein and inhibits K48-linked polyubiquitination, leading to YAP stabilization

  • RNF181 depletion decreases YAP protein levels and Hippo signaling target genes (CTGF and CYR61)

  • Its depletion significantly inhibits TNBC cell migration, invasion, and proliferation

These findings suggest that RNF181 employs non-proteolytic mechanisms to regulate cancer-associated proteins, enhancing their stability rather than promoting their degradation.

What prognostic significance does RNF181 expression have in breast cancer?

Analysis of public databases (Oncomine and KMPLOT) indicates that:

These findings suggest that RNF181 could serve as a prognostic marker in breast cancer generally, and in TNBC specifically. The correlation with YAP expression in TNBC further supports RNF181's role in modulating the Hippo/YAP pathway in these aggressive tumors.

What experimental approaches are used to study RNF181's effects on cancer cells?

Several complementary methodologies have been employed to investigate RNF181 in cancer:

• Expression analysis:

  • Western blot for protein quantification

  • Real-time PCR for target gene expression

  • Unbiased RNA sequencing for genome-wide expression analysis

• Functional assays:

  • WST1 assay for cell proliferation

  • Trans-well and wound healing assays for cell migration and invasion

  • In vivo tumor growth assays

• Molecular mechanism studies:

  • Protein stability and ubiquitin assays

  • Immunoprecipitation to detect protein interactions

  • Immunostaining for protein localization

  • Ubiquitin-based immunoprecipitation to detect specific ubiquitination patterns (K48 vs. K63-linked)

These methods collectively enable researchers to comprehensively assess RNF181's role in cancer progression from phenotypic effects to detailed molecular mechanisms.

How does RNF181 regulate NF-κB signaling in lymphocytes?

RNF181 functions as a negative regulator of antigen receptor signaling to NF-κB in lymphocytes through the following mechanism:

• RNF181 interacts with CARD11, a key signaling scaffold in the antigen receptor pathway

• It reduces the levels of Bcl10, an obligate signaling protein, even prior to signaling activation

• Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro

• Through this mechanism, RNF181 limits NF-κB activation downstream of antigen receptor engagement

This regulatory function has implications for both normal lymphocyte activation during immune responses and for pathological conditions where NF-κB signaling is dysregulated, such as in diffuse large B cell lymphoma (DLBCL).

What is the significance of RNF181 in lymphoma progression?

RNF181 has been shown to limit the proliferation of human diffuse large B cell lymphoma (DLBCL) cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture . This suggests that:

• RNF181 serves as a natural brake on excessive NF-κB activation

• It represents a regulatory checkpoint that can modulate the output of CARD11 signaling in both normal and transformed lymphocytes

• Modulating RNF181 activity might offer therapeutic opportunities for certain types of lymphoma where NF-κB signaling is constitutively active

Understanding these regulatory mechanisms could provide insights into developing targeted therapies for lymphomas with dysregulated NF-κB signaling.

What novel techniques have been developed to study RNF181 protein interactions?

Bioluminescence resonance energy transfer-based interaction cloning (BRIC) represents an innovative screening strategy developed specifically for studying CARD11 interactors, which led to the identification of RNF181 . This technique:

• Detects protein-protein interactions in live mammalian cells

• Functions in a high-throughput manner

• Uses CARD11ΔID-Rluc8 fusion protein as bait and YPet-tagged candidate interactors

• Defines positive interactions as those producing mBRET values at least 3-fold higher than bystander values

• Allows for the purification of interacting proteins by sib selection and sequencing

This methodology offers advantages over traditional yeast two-hybrid or pull-down approaches by detecting interactions in a native cellular context with proper post-translational modifications and subcellular localization.

How can researchers distinguish between direct and indirect RNF181 interactions?

Distinguishing between direct protein-protein interactions and indirect associations mediated by nucleic acids (RNA/DNA) requires specialized methodologies:

• RNase/DNase treatment before immunoprecipitation:

  • In studies of HERC2 (another E3 ligase) in hESCs, RNase A treatment prior to immunoprecipitation reduced the number of significant interactors from 113 to 33 proteins

  • This approach can be applied to RNF181 studies to determine RNA-dependent interactions

• In vitro binding assays with purified proteins:

  • Using recombinant proteins to verify direct interactions

  • This approach was used to confirm that Bcl10 can serve as a direct substrate for RNF181 E3 ligase activity

• Domain mapping and mutational analysis:

  • Creating truncations and point mutations in RNF181 or its potential partners

  • Identifying specific domains required for interaction

These complementary approaches help researchers build confidence in identifying genuine direct interactors of RNF181 versus those associated through larger complexes.

What are the technical challenges in studying RNF181-mediated ubiquitination?

Studying RNF181-mediated ubiquitination presents several technical challenges that researchers must address:

• Distinguishing ubiquitination patterns:

  • K48-linked polyubiquitination (typically signals for proteasomal degradation)

  • K63-linked polyubiquitination (often involved in protein stabilization or signaling)

  • This requires ubiquitin-based immunoprecipitation assays with linkage-specific antibodies

• Functional redundancy among E3 ligases:

  • Multiple E3 ligases may have overlapping substrates

  • RNF181 knockdown studies show lack of strong phenotypes in some contexts, suggesting compensatory mechanisms

  • Studies have observed multiple common interactors between distinct E3 enzymes

• Distinguishing direct versus indirect effects of RNF181 depletion:

  • Only a marginal number of RNF181 interacting partners increase upon its knockdown

  • RNF181 may regulate multiple processes in proteasome-independent manners

Addressing these challenges requires combining genetic approaches (knockdowns, knockouts), biochemical assays (ubiquitination assays with various ubiquitin mutants), and systems biology approaches (proteome-wide analyses).

How can cell type-specific functions of RNF181 be systematically investigated?

The search results demonstrate that RNF181 has significantly different interaction partners and functions across cell types. For instance, only 17% of RNF181 interactors in hESCs remain significant interactors in neuronal cells . To systematically investigate these cell type-specific functions, researchers should consider:

• Comparative interactomics:

  • Perform parallel immunoprecipitation-mass spectrometry studies across multiple cell types

  • Use identical experimental conditions to allow direct comparison

  • Apply computational approaches to identify cell type-specific versus common interactors

• Single-cell analysis:

  • Employ single-cell proteomics or transcriptomics to assess RNF181 function in heterogeneous populations

  • This could reveal cell state-dependent roles of RNF181 even within nominally similar cell types

• Tissue-specific conditional knockout models:

  • Generate conditional RNF181 knockout models to assess tissue-specific functions in vivo

  • This would overcome potential developmental defects from global knockout

Understanding these cell type-specific functions could reveal specialized roles of RNF181 in normal development and disease contexts.

What are the therapeutic implications of targeting RNF181 in cancer?

Based on the findings that RNF181 promotes cancer progression in different breast cancer subtypes, there are several therapeutic considerations:

• Potential for dual targeting in different breast cancer subtypes:

  • In ERα-positive breast cancer: Targeting RNF181 could reduce ERα stability and signaling

  • In TNBC: Inhibiting RNF181 could destabilize YAP, reducing Hippo pathway activation

  • This suggests RNF181 inhibition might be effective across breast cancer subtypes

• Addressing endocrine resistance:

  • RNF181 expression correlates with poor survival in endocrine-treated patients

  • Targeting RNF181 might help overcome endocrine resistance in ER-positive breast cancer

• Development of specific inhibitors:

  • As an E3 ubiquitin ligase, RNF181 contains a RING finger domain that could be targeted

  • Small molecule inhibitors disrupting specific protein-protein interactions (e.g., RNF181-ERα or RNF181-YAP) might offer more selective approaches

• Considerations of toxicity:

  • Given RNF181's roles in normal stem cells and lymphocytes, potential side effects need careful evaluation

  • Tissue-specific delivery strategies might be necessary

Detailed understanding of RNF181's substrate specificity and regulatory mechanisms will be essential for developing effective therapeutic strategies.

How might RNF181 function interact with other post-translational modifications?

While the search results focus on RNF181's role in protein ubiquitination, a comprehensive understanding would require investigating interactions with other post-translational modifications:

• Phosphorylation interplay:

  • Many E3 ligases recognize phosphorylated substrates

  • The Hippo/YAP pathway is heavily regulated by phosphorylation

  • Research should investigate whether RNF181 preferentially recognizes phosphorylated forms of YAP or other substrates

• SUMOylation crosstalk:

  • SUMO modification often competes with ubiquitination for the same lysine residues

  • Investigating potential crosstalk between RNF181-mediated ubiquitination and SUMOylation of substrates could reveal additional regulatory mechanisms

• Acetylation interactions:

  • Acetylation of lysine residues can prevent ubiquitination

  • ERα is known to be regulated by acetylation

  • Studies could examine how acetylation affects RNF181-mediated stabilization of ERα

Understanding these complex interactions between different post-translational modification systems would provide a more complete picture of RNF181's regulatory functions.