EXOSC10 Human

What is EXOSC10 and what are its primary functions in human cells?

EXOSC10, also known as PM/Scl-100 in humans (or Rrp6 in yeast and fly), is a 3′–5′ exoribonuclease that functions as a catalytic subunit of the multimeric nuclear RNA exosome complex . The protein plays essential roles in multiple RNA processing pathways, including:

• Processing biologically active transcripts

• Degrading aberrant mRNAs

• Targeting specific long non-coding RNAs (lncRNAs)

• Regulating Xist RNA and the onset of X chromosome inactivation in somatic cells

EXOSC10 can function both as part of the exosome complex and independently, as it has been found to bind RNA directly through its C-terminal domain . Its conservation across species underscores its fundamental importance in RNA metabolism across eukaryotes.

How does EXOSC10 differ structurally and functionally from other exosome components?

EXOSC10 stands apart from other exosome components due to its catalytic activity and specific structural domains. Unlike core exosome subunits, EXOSC10 possesses intrinsic 3′-5′ exoribonuclease activity that contributes to the processing capacity of the complex . The protein can also function independently of the exosome core subunits in some contexts .

A distinctive feature of EXOSC10 is its C-terminal RNA-binding domain, which enables direct interaction with RNA substrates . This domain is not present in all exosome components and contributes to EXOSC10's substrate specificity. While the exosome complex contains another catalytic subunit, DIS3, research has demonstrated that EXOSC10 and DIS3 have different functions, with EXOSC10 playing more critical roles in certain processes such as DNA repair by homologous recombination .

What RNA substrates are specifically targeted by EXOSC10 in human cells?

EXOSC10 processes multiple RNA substrate types in human cells with varying degrees of specificity. Research has identified several key targets:

• Aberrant mRNAs: EXOSC10 participates in surveillance mechanisms that identify and degrade defective messenger RNAs .

• Long non-coding RNAs (lncRNAs): Specific classes of lncRNAs are targeted by EXOSC10 for degradation or processing .

• rRNA processing intermediates: EXOSC10 plays a crucial role in ribosomal RNA maturation. Knockout studies have shown that EXOSC10 depletion leads to rRNA processing defects .

• Short poly(A) RNAs: Depletion of EXOSC10 in human cell lines stabilizes short poly(A) RNAs and increases the length of their poly(A) tails .

• Diluted lncRNAs (dilncRNAs): EXOSC10 depletion impacts exoribonucleolysis of nuclear RNA metabolism and can lead to increased levels of dilncRNAs .

EXOSC10's substrate specificity appears to be determined by both intrinsic sequence recognition and through interactions with cofactors that direct the enzyme to particular RNA targets.

How do we experimentally determine EXOSC10's role in transcriptome remodeling?

To investigate EXOSC10's function in transcriptome remodeling, researchers employ several complementary methodological approaches:

• Conditional knockout (cKO) models: Using CRISPR/Cas9-mediated gene editing to create tissue-specific or inducible EXOSC10 knockout, as demonstrated in mouse oocyte studies . This allows analysis of phenotypic changes and molecular consequences of EXOSC10 depletion.

• RNA-seq analysis following EXOSC10 depletion: Single-cell or bulk RNA sequencing can identify transcripts that accumulate or decrease upon EXOSC10 knockdown. This approach revealed that EXOSC10 shapes the transcriptome encoding endomembrane components and cell cycle regulators in mouse oocytes .

• Rescue experiments with wild-type and mutant EXOSC10: Establishing cell lines with tetracycline-inducible expression of siRNA-resistant EXOSC10 (both wild-type and mutant versions) enables functional testing of specific domains or modifications .

• Immunoprecipitation followed by RNA sequencing: This identifies direct RNA targets bound by EXOSC10 in vivo.

In one key study, researchers performed multiple single oocyte RNA-seq after EXOSC10 depletion, which documented dysregulation of several RNA types, particularly mRNAs encoding proteins important for endomembrane trafficking and meiotic cell cycle progression .

How does EXOSC10 contribute to DNA double-strand break repair in human cells?

EXOSC10 plays a critical and specific role in the homologous recombination (HR) pathway of DNA double-strand break (DSB) repair. According to detailed mechanistic studies, EXOSC10:

• Is recruited to DSB sites along with other exosome components like DIS3 .

• Functions at a specific step in the HR pathway, being required for proper recruitment of RPA (Replication Protein A) to DSBs . Depletion of EXOSC10 significantly reduces the percentage of RPA-positive DNA damage tracks (from 75.4% to 47.2%) .

• Acts downstream of CtIP (which initiates DNA end resection) but upstream of RPA and RAD51 assembly. EXOSC10 depletion impairs RAD51 recruitment to DSBs (reducing positive tracks from 35.6% to 12.6%) but does not affect CtIP recruitment .

• Regulates DNA end resection termination. While initial resection occurs normally in EXOSC10-depleted cells, the levels of single-stranded DNA (ssDNA) at distances further from the break (1500 and 3500 nucleotides) are significantly higher than in control cells, suggesting that EXOSC10 normally helps terminate the resection process .

The exosome's RNA processing activity at DNA damage sites appears to be necessary for proper coordination of the HR repair pathway, with EXOSC10 being the crucial catalytic subunit in this process.

What experimental methods are most effective for studying EXOSC10's function in DNA repair?

To investigate EXOSC10's role in DNA repair, researchers employ several specialized techniques:

• Micro-irradiation combined with immunostaining: This approach involves using laser micro-irradiation to induce localized DNA damage in cellular nuclei, followed by immunofluorescence to detect recruitment of repair proteins. This method effectively demonstrated that EXOSC10 depletion impairs the recruitment of RAD51 and RPA to damage sites .

• Single-Molecule Analysis of Resection Tracks (SMART): This technique measures the length of resected DNA at break sites and revealed that EXOSC10 depletion results in significantly longer ssDNA tracks compared to control cells .

• Quantitative resection assay using site-specific DSBs: This method uses the DIvA (DSB-induced via AsiSI) cell system, where the AsiSI restriction enzyme is fused with an estrogen receptor domain that translocates to the nucleus upon 4-hydroxytamoxifen treatment. Combined with qPCR analysis, this approach can quantify ssDNA at sequence-specific DSBs and at different distances from the break site .

• Ionizing radiation-induced foci (IRIF) analysis: This assay examines the formation of repair protein foci after exposure to ionizing radiation and showed that EXOSC10 depletion inhibits the assembly of RPA foci in irradiated cells .

• siRNA knockdown followed by functional assays: Depleting EXOSC10 using siRNA and then measuring HR efficiency using reporter constructs helps establish the functional significance of EXOSC10 in the repair process.

How do post-translational modifications affect EXOSC10 function?

EXOSC10 undergoes several post-translational modifications that significantly impact its function, localization, and stability:

• SUMOylation: EXOSC10 can be SUMOylated at lysine residue K583, which affects its function. The ubiquitin-specific protease USP36 interacts with EXOSC10 and influences this SUMOylation process . Researchers have developed EXOSC10_K583R mutants to study the functional consequences of preventing this modification .

• Regulation during cell cycle progression: In germ cells, EXOSC10 undergoes post-translational regulation during meiosis. The protein becomes unstable at later stages of gamete development, similar to how its yeast orthologue Rrp6 becomes unstable during meiosis . This regulated instability may be important for proper germ cell differentiation.

• Nucleolar-cytoplasmic distribution: Post-translational modifications likely regulate EXOSC10's localization between nucleoli and the cytoplasm of mitotic and meiotic germ cells .

To experimentally investigate these modifications, researchers have developed specialized tools including:

• Mutation of specific residues (e.g., K583R) to prevent modification

• Generation of tetracycline-inducible expression systems for wild-type and mutant forms

• Production of recombinant proteins using bacterial expression systems with tags like GST-EXOSC10 fusion proteins

What is the significance of EXOSC10's association with the XY body during meiosis?

EXOSC10 exhibits a fascinating temporal association with the XY body during male meiosis, which has important implications for understanding its role in epigenetic regulation:

• Transient XY body association: Research has demonstrated that EXOSC10 transiently associates with the XY body, a specialized chromatin structure formed by the X and Y chromosomes during male meiosis .

• Connection to meiotic sex chromosome inactivation (MSCI): The XY body is targeted by meiotic sex chromosome inactivation (MSCI), a process that silences the X and Y chromosomes during male meiosis. EXOSC10's association with this structure suggests potential involvement in this epigenetic silencing process .

• Parallel to X chromosome inactivation: This finding is particularly significant when considered alongside evidence that EXOSC10 regulates Xist RNA and the onset of X chromosome inactivation in somatic cells , suggesting conserved functions in different types of sex chromosome silencing.

• Potential role in chromatin remodeling: The association of an RNA processing enzyme with a specialized chromatin domain suggests potential functions in RNA-mediated chromatin remodeling during meiosis.

Conditional knockout studies in male germ cells using Cre recombinase controlled by Stra8 or Ddx4/Vasa promoters have shown that EXOSC10-deficient mice have small testes, impaired germ cell differentiation, and are subfertile , supporting its essential role in male germline development.

What evidence supports EXOSC10 as a potential biomarker for hepatocellular carcinoma?

Comprehensive bioinformatics analysis and experimental verification have identified EXOSC10 as a novel prognostic biomarker for hepatocellular carcinoma (HCC), with several lines of evidence supporting this role:

• Unfavorable prognosis correlation: Recent evidence indicates that human EXOSC10 is an unfavorable prognostic marker for liver cancer . Higher expression levels correlate with worse clinical outcomes.

• Functional relevance to cancer biology: EXOSC10's roles in RNA metabolism and transcriptional control are processes frequently dysregulated in cancer. Its depletion affects exoribonucleolysis of nuclear RNA metabolism and transcriptional control, potentially influencing cancer cell behavior .

• Potential therapeutic target: Given the poor prognosis for HCC patients (with around 70% relapsing within five years after operation ), identifying novel biomarkers like EXOSC10 could guide the development of targeted therapies.

The research on EXOSC10 as an HCC biomarker addresses a critical clinical need, as hepatocellular carcinoma represents 4.7% of new cancer cases globally, with 830,180 deaths (8.3%) in 2020 . Current treatment methods include local radio frequency ablation, partial hepatectomy, and liver transplantation, but long-term outcomes remain poor .

How might EXOSC10's role in DNA repair contribute to cancer development or treatment?

EXOSC10's crucial involvement in homologous recombination repair of DNA double-strand breaks has significant implications for cancer:

• Potential contributor to genomic instability: Defects in EXOSC10 function could lead to improper DNA repair, resulting in genomic instability—a hallmark of cancer. The specific defects observed in EXOSC10-depleted cells include:

  • Impaired RPA recruitment to DNA damage sites (reduced from 75.4% to 47.2% positive tracks)

  • Defective RAD51 filament formation (reduced from 35.6% to 12.6% positive tracks)

  • Dysregulated DNA end resection termination

• Synthetic lethality opportunities: Cancer cells with mutations in certain DNA repair pathways may become more dependent on EXOSC10-mediated repair functions, creating opportunities for synthetic lethality approaches in cancer therapy.

• Relationship to DNA repair-targeted treatments: Many cancer therapies, including radiation and certain chemotherapeutics, work by inducing DNA damage. The effectiveness of these treatments might be influenced by EXOSC10 expression or activity levels in tumor cells.

• Biomarker potential for therapy response: EXOSC10 expression or activity could potentially serve as a biomarker for predicting response to DNA-damaging therapies or PARP inhibitors, which are particularly effective against tumors with defects in homologous recombination.

Experimental approaches to further explore these connections could include analyzing EXOSC10 expression in tumors with different DNA repair deficiencies, testing EXOSC10 inhibition in combination with standard DNA-damaging agents, and examining correlations between EXOSC10 expression and therapy response in patient samples.

What are the most effective experimental systems for investigating EXOSC10 function in humans?

Several experimental systems have proven particularly effective for studying EXOSC10 function in human contexts:

• Human cell line models with inducible expression systems:

  • HeLa-TR cells with tetracycline-inducible expression of siRNA-resistant EXOSC10 (wild-type and mutant versions)

  • DIvA (DSB-induced via AsiSI) cells for studying DNA repair functions

  • These systems allow temporal control of EXOSC10 expression and the ability to perform rescue experiments with mutant forms

• CRISPR/Cas9-mediated gene editing approaches:

  • While complete knockout may be lethal in some systems, conditional knockout approaches can be highly informative

  • Domain-specific mutations can be introduced to test the importance of specific functional regions

• Recombinant protein expression systems:

  • GST-EXOSC10 fusion proteins expressed in bacterial systems

  • His-tagged protein purification for in vitro biochemical assays

  • These systems enable detailed structure-function analyses

• Specialized assays for specific EXOSC10 functions:

  • SMART (Single-Molecule Analysis of Resection Tracks) for DNA repair studies

  • RNA-seq following EXOSC10 depletion to identify regulated transcripts

  • Immunoprecipitation combined with mass spectrometry to identify interaction partners

• Patient-derived materials:

  • Samples from patients with polymyositis/scleroderma overlap syndrome, where EXOSC10 is a target of autoantibodies

  • Hepatocellular carcinoma tissue for biomarker evaluation

Each system has particular strengths depending on the specific aspect of EXOSC10 biology being investigated, and combining multiple approaches provides the most comprehensive understanding.

What are the key considerations when designing knockdown or knockout strategies for EXOSC10?

Designing effective knockdown or knockout strategies for EXOSC10 requires careful consideration of several factors:

• Complete knockout lethality: Given EXOSC10's essential functions, complete knockout may be lethal in many systems. Researchers should consider:

  • Conditional knockout systems (tissue-specific or inducible)

  • Partial knockdown approaches

  • Rescue experiments with wild-type or mutant proteins

• siRNA design and validation:

  • Validated siRNA sequences: For example, the sequence 5′-GGATGAGTCCTACCTTGAA-3′ has been successfully used to target EXOSC10

  • Controls for off-target effects

  • Confirmation of knockdown efficiency at both mRNA and protein levels

• CRISPR/Cas9 considerations:

  • Guide RNA design to minimize off-target effects

  • Selection of appropriate promoters for tissue-specific expression (e.g., Stra8 or Ddx4/Vasa promoters for germ cell-specific expression)

  • Verification of indel formation and frameshift mutations

• Rescue system development:

  • Creation of siRNA-resistant EXOSC10 constructs through silent mutations in the siRNA target sequence (e.g., 5′-GGACGAATCTTATTTGGAA-3′)

  • Tetracycline-inducible expression systems for controlled rescue experiments

  • Inclusion of wild-type and mutant versions (e.g., K583R) to test specific functions

• Phenotypic analysis timepoints:

  • Consideration of EXOSC10's role in both acute processes (e.g., DNA repair) and more long-term functions (e.g., RNA metabolism)

  • Selection of appropriate timepoints post-depletion for analyzing different processes

A successful example is the oocyte-specific conditional knockout of Exosc10 in mice using CRISPR/Cas9, which resulted in female subfertility due to delayed germinal vesicle breakdown, allowing researchers to study its function in germ cell development while avoiding embryonic lethality .

What are the emerging connections between EXOSC10 and cellular stress responses?

Emerging research suggests significant connections between EXOSC10 and various cellular stress response pathways, opening new avenues for investigation:

• DNA damage response integration: EXOSC10's critical role in homologous recombination repair suggests it may serve as an integrator between RNA processing and DNA damage responses . The protein's recruitment to DNA break sites and its requirement for proper RPA assembly point to a coordination mechanism between these fundamental cellular processes.

• Cell cycle checkpoint regulation: Evidence from mouse oocyte studies indicates that EXOSC10 depletion affects CDK1 activation, possibly due to persistent WEE1 activity, which blocks lamina phosphorylation and disassembly . This suggests EXOSC10 may influence cell cycle checkpoints during stress conditions.

• Endomembrane system remodeling during stress: EXOSC10 shapes the transcriptome encoding endomembrane components (endosomes, Golgi, ER, lysosomes, autophagosomes) , systems that are extensively remodeled during various cellular stresses. The protein may help coordinate membrane trafficking adaptations to stress conditions.

• rRNA processing and nucleolar stress: EXOSC10 depletion causes rRNA processing defects , potentially triggering nucleolar stress responses. The nucleolus serves as a stress sensor in cells, and EXOSC10's nucleolar localization in certain cell types suggests potential involvement in nucleolar stress signaling.

Future research could focus on how EXOSC10 activity is modulated during different stress conditions and whether its RNA processing functions are redirected toward specific stress-responsive transcripts.

How might therapeutic targeting of EXOSC10 be developed for cancer treatment?

Developing therapeutic approaches targeting EXOSC10 for cancer treatment presents both opportunities and challenges that warrant sophisticated research strategies:

• Selective inhibition approaches:

  • Development of small molecule inhibitors targeting EXOSC10's catalytic domain

  • Exploration of allosteric modulators that affect EXOSC10's interaction with the core exosome or specific substrates

  • Investigation of compounds that disrupt the SUMOylation of EXOSC10 at K583, which affects its function

• Cancer-specific vulnerabilities:

  • Identification of cancer types with heightened dependence on EXOSC10 function

  • Exploration of synthetic lethality approaches, particularly in cancers with defects in complementary DNA repair pathways

  • Investigation of how EXOSC10 inhibition might sensitize cancer cells to existing therapies, particularly DNA-damaging agents

• Biomarker-guided therapy:

  • Utilization of EXOSC10 as a prognostic biomarker in hepatocellular carcinoma

  • Development of companion diagnostics to identify patients most likely to benefit from EXOSC10-targeted therapies

  • Integration of EXOSC10 expression data with other molecular profiles to guide personalized treatment approaches

• Delivery challenges and solutions:

  • Development of targeted delivery systems to limit inhibition to cancer cells

  • Exploration of cancer-specific promoters for RNA interference approaches

  • Investigation of proteasome-targeting chimeras (PROTACs) to achieve selective EXOSC10 degradation in cancer cells

• Mitigating potential toxicities:

  • Development of partial inhibitors that reduce but do not eliminate EXOSC10 function

  • Pulsed dosing strategies to allow recovery of essential EXOSC10 functions in normal cells

  • Identification of cancer-specific EXOSC10 dependencies that differ from those in normal cells