The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.

Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile.

Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications; however, these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in an HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme-mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.

Dysregulation of RNA-Exosome machinery is directly linked to major cancer hallmarks in prostate cancer: Oncogenic role of PABPN1.

Novel biomarkers and therapeutic strategies for prostate-cancer (PCa) are required to overcome its lethal progression. The dysregulation/implication of the RNA-Exosome-complex (REC; cellular machinery controlling the 3'-5'processing/degradation of most RNAs) in different cancer-types, including PCa, is poorly known. Herein, different cellular/molecular/preclinical approaches with human PCa-samples (tissues and/or plasma of 7 independent cohorts), and in-vitro/in-vivo PCa-models were used to comprehensively characterize the REC-profile and explore its role in PCa. Moreover, isoginkgetin (REC-inhibitor) effects were evaluated on PCa-cells. We demonstrated a specific dysregulation of the REC-components in PCa-tissues, identifying the Poly(A)-Binding-Protein-Nuclear 1 (PABPN1) factor as a critical regulator of major cancer hallmarks. PABPN1 is consistently overexpressed in different human PCa-cohorts and associated with poor-progression, invasion and metastasis. PABPN1 silencing decreased relevant cancer hallmarks in multiple PCa-models (proliferation/migration/tumourspheres/colonies, etc.) through the modulation of key cancer-related lncRNAs (PCA3/FALEC/DLEU2) and mRNAs (CDK2/CDK6/CDKN1A). Plasma PABPN1 levels were altered in patients with metastatic and tumour-relapse. Finally, pharmacological inhibition of REC-activity drastically inhibited PCa-cell aggressiveness. Altogether, the REC is drastically dysregulated in PCa, wherein this novel molecular event/mechanism, especially PABPN1 alteration, may be potentially exploited as a novel prognostic and therapeutic tool for PCa.

EXOSC5 maintains cancer stem cell activity in endometrial cancer by regulating the NTN4/integrin ß1 signalling axis.

Endometrial carcinoma (EC) is a common type of uterine cancer in developed countries, originating from the uterine epithelium. The incidence rate of EC in Taiwan has doubled from 2005. Cancer stem cells (CSCs) are a subpopulation of cancer cells that have high tumorigenicity and play a crucial role in the malignant processes of cancer. Targeting molecules associated with CSCs is essential for effective cancer treatments. This study delves into the role of Exosome component 5 (EXOSC5) in EC. Data from The Cancer Genome Atlas suggests a correlation between high EXOSC5 mRNA expression and unfavorable EC prognosis. EXOSC5 knockdown diminished EC-CSC self-renewal and reduced expression of key cancer stemness proteins, including c-MYC and SOX2. Intriguingly, this knockdown significantly curtailed tumorigenicity and CSC frequency in EC tumor spheres. A mechanistic examination revealed a reduction in netrin4 (NTN4) levels in EXOSC5-depleted EC cells. Moreover, NTN4 treatment amplified EC cell CSC activity and, when secreted, NTN4 partnered with integrin ß1, subsequently triggering the FAK/SRC axis to elevate c-MYC activity. A clear positive relation between EXOSC5 and NTN4 was evident in 93 EC tissues. In conclusion, EXOSC5 augments NTN4 expression, activating c-MYC via the integrin ß1/FAK/SRC pathway, offering potential avenues for EC diagnosis and treatment.

Effect of RNA-binding proteins on osteogenic differentiation of bone marrow mesenchymal stem cells.

Tissue regeneration mediated by mesenchymal stem cells (MSCs) is an ideal way to repair bone defects. RNA-binding proteins (RBPs) can affect cell function through post-transcriptional regulation. Exploring the role of RBPs in the process of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is helpful to find a key method to promote the osteogenic efficiency of BMSCs. By reviewing the literature, we obtained a differentially expressed mRNA dataset during the osteogenic differentiation of BMSCs and a human RBP dataset. A total of 82 differentially expressed RBPs in the osteogenic differentiation of BMSCs were screened by intersection of the two datasets. Functional analysis showed that the differentially expressed RBPs were mainly involved in RNA transcription, translation and degradation through the formation of spliceosomes and ribonucleoprotein complexes. The top 15 RBPs determined by degree score were FBL, NOP58, DDX10, RPL9, SNRPD3, NCL, IFIH1, RPL18A, NAT10, EXOSC5, ALYREF, PA2G4, EIF5B, SNRPD1 and EIF6. The results of this study demonstrate that the expression of many RBPs changed during osteogenic differentiation of BMSCs.

DIS3 depletion in multiple myeloma causes extensive perturbation in cell cycle progression and centrosome amplification.

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

[Expression and Clinical Significance of Exosome Component 4 in Newly Diagnosed Patients with Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To explore the expression of Exosome Component 4（EXOSC4） in the tissues of newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) and its clinical significance. METHODS: The expression of EXOSC4 protein in the tissues of 181 newly diagnosed DLBCL patients was analyzed by immunohistochemical staining. Clinical data were collected. The correlation between EXOSC4 protein expression in the tissues of newly diagnosed DLBCL patients and clinical features were analyzed and its prognostic significance. RESULTS: The positive rate of EXOSC4 protein expression was 68.51% in the tissues of 181 newly diagnosed DLBCL patients. These patients were divided into two groups, with 44 cases in high expression group and 137 cases in low expression group. There were no significant differences in age, gender, B symptoms, serum lactate dehydrogenase (LDH) level, Eastern Cooperative Oncology Group (ECOG) score, Ann Arbor stage, extranodal disease, International Prognostic Index (IPI) score, National Comprehensive Cancer Network IPI (NCCN-IPI) score, and cell origin between the two groups (P>0.05). Cox multivariate regression analysis showed that high EXOSC4 protein expression in tissues was an independent poor prognostic factor for OS and PFS in newly diagnosed DLBCL patients (all P<0.05). K-M survival analysis showed that newly diagnosed DLBCL patients with high EXOSC4 protein expression had significantly shorter overall survival (OS) and progression free survival (PFS) than those patients with low EXOSC4 protein expression (all P<0.05). CONCLUSION: High EXOSC4 protein expression in tissues of newly diagnosed DLBCL patients is an independent poor prognostic factor for survival.

Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

Long, noncoding RNAs (lncRNAs) are indispensable for normal cell physiology and, consequently, are tightly regulated in human cells. Yet, unlike mRNA, substantially less is known about the mechanisms for lncRNA degradation. It is important to delineate the regulatory control of lncRNA degradation, particularly for lncRNA telomeric repeat-containing RNA (TERRA), as the TERRA-telomere R-loops dictate cell cycle progression and genomic stability. We now report that the exosome complex component Exosc9 degrades lncRNA TERRA in human mammary epithelial cells. Heterochromatin protein 1 alpha (HP1α) recruits Exosc9 to the telomeres; specifically, the SUMO-modified form of HP1α supports interaction with Exosc9 and, as previously reported, lncRNA TERRA. The telomeric enrichment of Exosc9 is cell cycle-dependent and consistent with the loss of telomeric TERRA in the S/G2 phase. Elevated Exosc9 is frequently observed and drives the growth of endocrine therapy-resistant (ET-R) HR+ breast cancer (BCa) cells. Specifically, the knockdown of Exosc9 inversely impacts telomeric R-loops and the integrity of the chromosome ends of ET-R cells. Consistently, Exosc9 levels dictate DNA damage and the sensitivity of ET-R BCa cells to PARP inhibitors. In this regard, Exosc9 may serve as a promising biomarker for predicting the response to PARP inhibitors as a targeted monotherapy for ET-R HR+ BCa.

Unfolded protein response pathways in stroke patients: a comprehensive landscape assessed through machine learning algorithms and experimental verification.

BACKGROUND: The unfolding protein response is a critical biological process implicated in a variety of physiological functions and disease states across eukaryotes. Despite its significance, the role and underlying mechanisms of the response in the context of ischemic stroke remain elusive. Hence, this study endeavors to shed light on the mechanisms and role of the unfolding protein response in the context of ischemic stroke. METHODS: In this study, mRNA expression patterns were extracted from the GSE58294 and GSE16561 datasets in the GEO database. The screening and validation of protein response-related biomarkers in stroke patients, as well as the analysis of the immune effects of the pathway, were carried out. To identify the key genes in the unfolded protein response, we constructed diagnostic models using both random forest and support vector machine-recursive feature elimination methods. The internal validation was performed using a bootstrapping approach based on a random sample of 1,000 iterations. Lastly, the target gene was validated by RT-PCR using clinical samples. We utilized two algorithms, CIBERSORT and MCPcounter, to investigate the relationship between the model genes and immune cells. Additionally, we performed uniform clustering of ischemic stroke samples based on expression of genes related to the UPR pathway and analyzed the relationship between different clusters and clinical traits. The weighted gene co-expression network analysis was conducted to identify the core genes in various clusters, followed by enrichment analysis and protein profiling for the hub genes from different clusters. RESULTS: Our differential analysis revealed 44 genes related to the UPR pathway to be statistically significant. The integration of both machine learning algorithms resulted in the identification of 7 key genes, namely ATF6, EXOSC5, EEF2, LSM4, NOLC1, BANF1, and DNAJC3. These genes served as the foundation for a diagnostic model, with an area under the curve of 0.972. Following 1000 rounds of internal validation via randomized sampling, the model was confirmed to exhibit high levels of both specificity and sensitivity. Furthermore, the expression of these genes was found to be linked with the infiltration of immune cells such as neutrophils and CD8 T cells. The cluster analysis of ischemic stroke samples revealed three distinct groups, each with differential expression of most genes related to the UPR pathway, immune cell infiltration, and inflammatory factor secretion. The weighted gene co-expression network analysis showed that all three clusters were associated with the unfolded protein response, as evidenced by gene enrichment analysis and the protein landscape of each cluster. The results showed that the expression of the target gene in blood was consistent with the previous analysis. CONCLUSION: The study of the relationship between UPR and ischemic stroke can help to better understand the underlying mechanisms of the disease and provide new targets for therapeutic intervention. For example, targeting the UPR pathway by blocking excessive autophagy or inducing moderate UPR could potentially reduce tissue injury and promote cell survival after ischemic stroke. In addition, the results of this study suggest that the use of UPR gene expression levels as biomarkers could improve the accuracy of early diagnosis and prognosis of ischemic stroke, leading to more personalized treatment strategies. Overall, this study highlights the importance of the UPR pathway in the pathology of ischemic stroke and provides a foundation for future studies in this field.

EXOSC10 is a novel hepatocellular carcinoma prognostic biomarker: a comprehensive bioinformatics analysis and experiment verification.

Background: Hepatocellular carcinoma (HCC) is a common malignant tumor. There are few studies on EXOSC10 (exosome component 10) in HCC; however, the importance of EXOSC10 for HCC remains unclear. Methods: In the study, the prognosis value of EXOSC10 and the immune correlation were explored by bioinformatics. The expression of EXOSC10 was verified by tissue samples from clinical patients and in vitro experiment (liver cancer cell lines HepG2, MHCC97H and Huh-7; normal human liver cell line LO2). Immunohistochemistry (IHC) was used to detect EXOSC10 protein expression in clinical tissue from HCC. Huh-7 cells with siEXOSC10 were constructed using lipofectamine 3000. Cell counting kit 8 (CCK-8) and colony formation were used to test cell proliferation. The wound healing and transwell were used to analyze the cell migration capacity. Mitochondrial membrane potential, Hoechst 33342 dye, and flow cytometer were used to detect the change in cell apoptosis, respectively. Differential expression genes (DEGs) analysis and gene set enrichment analysis (GSEA) were used to investigate the potential mechanism of EXOSC10 and were verified by western blotting. Results: EXOSC10 was highly expressed in tissues from patients with HCC and was an independent prognostic factor for overall survival (OS) in HCC. Increased expression of EXOSC10 was significantly related to histological grade, T stage, and pathological stage. Multivariate analysis indicated that the high expression level of EXOSC10 was correlated with poor overall survival (OS) in HCC. GO and GSEA analysis showed enrichment of the cell cycle and p53-related signaling pathway. Immune analysis showed that EXOSC10 expression was a significant positive correlation with immune infiltration in HCC. In vitro experiments, cell proliferation and migration were inhibited by the elimination of EXOSC10. Furthermore, the elimination of EXOSC10 induced cell apoptosis, suppressed PARP, N-cadherin and Bcl-2 protein expression levels, while increasing Bax, p21, p53, p-p53, and E-cadherin protein expression levels. Conclusions: EXOSC10 had a predictive value for the prognosis of HCC and may regulate the progression of HCC through the p53-related signaling pathway.

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii.

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii, an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3'-5' exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5'-3' exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identified in the archaeon Haloferax volcanii, which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3'-5' exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5'-3' exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

In vivo characterization of the critical interaction between the RNA exosome and the essential RNA helicase Mtr4 in Saccharomyces cerevisiae.

The RNA exosome is a conserved molecular machine that processes/degrades numerous coding and non-coding RNAs. The 10-subunit complex is composed of three S1/KH cap subunits (human EXOSC2/3/1; yeast Rrp4/40/Csl4), a lower ring of six PH-like subunits (human EXOSC4/7/8/9/5/6; yeast Rrp41/42/43/45/46/Mtr3), and a singular 3'-5' exo/endonuclease DIS3/Rrp44. Recently, several disease-linked missense mutations have been identified in structural cap and core RNA exosome genes. In this study, we characterize a rare multiple myeloma patient missense mutation that was identified in the cap subunit gene EXOSC2. This missense mutation results in a single amino acid substitution, p.Met40Thr, in a highly conserved domain of EXOSC2. Structural studies suggest that this Met40 residue makes direct contact with the essential RNA helicase, MTR4, and may help stabilize the critical interaction between the RNA exosome complex and this cofactor. To assess this interaction in vivo, we utilized the Saccharomyces cerevisiae system and modeled the EXOSC2 patient mutation into the orthologous yeast gene RRP4, generating the variant rrp4-M68T. The rrp4-M68T cells show accumulation of certain RNA exosome target RNAs and show sensitivity to drugs that impact RNA processing. We also identified robust negative genetic interactions between rrp4-M68T and specific mtr4 mutants. A complementary biochemical approach revealed that Rrp4 M68T shows decreased interaction with Mtr4, consistent with these genetic results. This study suggests that the EXOSC2 mutation identified in a multiple myeloma patient impacts the function of the RNA exosome and provides functional insight into a critical interface between the RNA exosome and Mtr4.

DIS3: The Enigmatic Gene in Multiple Myeloma.

Recent studies have revealed the genetic aberrations involved in the initiation and progression of various cancers, including multiple myeloma (MM), via next-generation sequencing analysis. Notably, DIS3 mutations have been identified in approximately 10% of patients with MM. Moreover, deletions of the long arm of chromosome 13, that includes DIS3, are present in approximately 40% of patients with MM. Regardless of the high incidence of DIS3 mutations and deletions, their contribution to the pathogenesis of MM has not yet been determined. Herein, we summarize the molecular and physiological functions of DIS3, focusing on hematopoiesis, and discuss the characteristics and potential roles of DIS3 mutations in MM. Recent findings highlight the essential roles of DIS3 in RNA homeostasis and normal hematopoiesis and suggest that the reduced activity of DIS3 may be involved in myelomagenesis by increasing genome instability.

RNA-regulatory exosome complex suppresses an apoptotic program to confer erythroid progenitor cell survival in vivo.

The RNA-regulatory exosome complex (EC) posttranscriptionally and cotranscriptionally processes and degrades RNAs in a context-dependent manner. Although the EC functions in diverse cell types, its contributions to stem and progenitor cell development are not well understood. Previously, we demonstrated that the transcriptional regulator of erythrocyte development, GATA1, represses EC subunit genes, and the EC maintains erythroid progenitors in vitro. To determine if this mechanism operates in vivo, we used the hematopoietic-specific Vav1-Cre and "conditional by inversion" mouse system to ablate Exosc3, encoding an EC structural subunit. Although Exosc3C/C Cre+ embryos developed normally until embryonic day 14.5, Exosc3 ablation was embryonic lethal and severely reduced erythromyeloid progenitor activity. RNA sequencing analysis of Exosc3-ablated burst-forming unit-erythroid revealed elevated transcripts encoding multiple proapoptotic factors, and the mutant erythroid progenitors exhibited increased apoptosis. We propose that the EC controls an ensemble of apoptosis-regulatory RNAs, thereby promoting erythroid progenitor survival and developmental erythropoiesis in vivo.

EXOSC8 promotes colorectal cancer tumorigenesis via regulating ribosome biogenesis-related processes.

Extensive protein synthesis is necessary for uncontrolled cancer cell proliferation, requiring hyperactive ribosome biogenesis. Our previous Pan-cancer study has identified EXOSC8 as a potential copy number variation (CNV)-driven rRNA metabolism-related oncogene in colorectal cancer (CRC). Herein, we further investigated proliferation-prompting functions and mechanisms of EXOSC8 in CRC by performing in silico analyses and wet-lab experiments. We uncovered that increased EXOSC8 expression and CNV levels are strongly associated with ribosome biogenesis-related factor levels in CRC, including ribosome proteins (RPs), eukaryotic translation initiation factors and RNA polymerase I/III. EXOSC8 silence decreases nucleolar protein and proliferation marker levels, as well as rRNA/DNA and global protein syntheses. Clinically, EXOSC8 is upregulated across human cancers, particularly CNV-driven upregulation in CRC was markedly associated with poor clinical outcomes. Mechanistically, EXOSC8 knockdown increased p53 levels in CRC, and the oncogenic proliferation phenotypes of EXOSC8 depended on p53 in vitro and in vivo. We discovered that EXOSC8 knockdown in CRC cells triggers ribosomal stress, nucleolar RPL5/11 being released into the nucleoplasm and "hijacking" Mdm2 to block its E3 ubiquitin ligase function, thus releasing and activating p53. Furthermore, our therapeutic experiments provided initial evidence that EXOSC8 might serve as a potential therapeutic target in CRC. Our findings revealed, for the first time, that the RNA exosome gene (EXOSC8) promotes CRC tumorigenesis by regulating cancer-related ribosome biogenesis in CRC. This study further extends our previous Pan-cancer study of the rRNA metabolism-related genes. The inhibition of EXOSC8 is a novel therapeutic strategy for the RPs-Mdm2-p53 ribosome biogenesis surveillance pathway in CRC.

MTR4 adaptor PICT1 functions in two distinct steps during pre-rRNA processing.

Ribosome biogenesis proceeds with the successive cleavage and trimming of the large 47S rRNA precursor, where the RNA exosome plays major roles in concert with the Ski2-like RNA helicase, MTR4. The recent finding of a consensus amino acid sequence, the arch-interacting motif (AIM), for binding to the arch domain in MTR4 suggests that recruitment of the RNA processing machinery to the maturing pre-rRNA at appropriate places and timings is mediated by several adaptor proteins possessing the AIM sequence. In yeast Saccharomyces cerevisiae, Nop53 plays such a role in the maturation of the 3'-end of 5.8S rRNA. Here, we investigated the functions of PICT1 (also known as GLTSCR2 or NOP53), a mammalian ortholog of Nop53, during ribosome biogenesis in human cells. PICT1 interacted with MTR4 and exosome in an AIM-dependent manner. Overexpression of PICT1 mutants defecting AIM sequence and siRNA-mediated depletion of PICT1 showed that PICT1 is involved in two distinct pre-rRNA processing steps during the generation of 60S ribosomes; first step is the early cleavage of 32S intermediate RNA, while the second step is the late maturation of 12S precursor into 5.8S rRNA. The recruitment of MTR4 and RNA exosome via the AIM sequence was required only during the late processing step. Although, the depletion of MTR4 and PICT1 induced stabilization of the tumor suppressor p53 protein in cancer cell lines, the depletion of the exosome catalytic subunits, RRP6 and DIS3, did not exert such an effect. These results suggest that recruitment of the RNA processing machinery to the 3'-end of pre-5.8S rRNA may be involved in the induction of the nucleolar stress response, but the pre-rRNA processing capabilities themselves were not involved in this process.

Integrated Bioinformatic Investigation of EXOSCs in Hepatocellular Carcinoma Followed by the Preliminary Validation of EXOSC5 in Cell Proliferation.

The Exosome complex (EXOSC) is a multiprotein complex that was originally discovered as the machinery of RNA degradation. Interestingly, recent studies have reported that EXOSC family members (EXOSCs) are associated with various human diseases, including cancers. It will be interesting to investigate whether EXOSCs are related to the processes of hepatocellular carcinoma (HCC). In this study, multiple public databases and experimental validation were utilized to systemically investigate the role of EXOSCs, especially EXOSC5, in HCC. It is worth considering that the mRNA and protein levels of many EXOSCs were elevated in HCC, although there were some differences in the results from different database analyses. The over-expression of EXOSCs could predict HCC to some extent, as evidenced by the positive correlation between the elevated EXOSCs and alpha fetoprotein (AFP) levels, as well as with a high accuracy, as shown by the receiver operating characteristic curve analysis. Additionally, higher mRNA expressions of specific EXOSCs were significantly related to clinical cancer stage, shorter overall survival and disease-free survival in HCC patients. A moderate mutation rate of EXOSCs was also observed in HCC. Furthermore, a gene functional enrichment analysis indicated that EXOSCs were mainly involved in the metabolism of RNA. Moreover, we revealed that the expression of EXOSCs is remarkably related to immune cell infiltration. Finally, EXOSC5 was upregulated in HCC tissues and cell lines, promoting cell growth and proliferation via activated signal transducer and activator of transcription 3 (STAT3). The bioinformatic analyses, following verification in situ and in vitro, provided a direction for further functions and underlying mechanism of EXOSCs in HCC.

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex.

RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined cryogenic electron microscopy structures of human nuclear exosome targeting (NEXT) complexes bound to RNA that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 surveying sequences upstream of the 3' end to facilitate RNA capture by MTR4. ZCCHC8 obscures MTR4 surfaces important for RNA binding and extrusion as well as MPP6-dependent recruitment and docking onto the RNA exosome core, interactions that contribute to RNA surveillance by coordinating RNA capture, translocation, and extrusion from the helicase to the exosome for decay.

Multiomics Integrated Analysis Identifies SLC24A2 as a Potential Link between Type 2 Diabetes and Cancer.

Background: So far, type 2 diabetes (T2D) is considered as an independent risk factor for various cancers, but the underlying mechanism remains unclear. Methods. SLC24A2 was first identified as a key gene strongly associated with fasting plasma glucose (FPG). Then, overlapped differentially expressed genes (DEGs) between T2D verse control and SLC24A2-high verse SLC24A2-low were extracted and imported into weighted correlation network analysis. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analysis were used for functional enrichment analysis of DEGs. Least absolute shrinkage and selection operator was utilized to build a T2D prediction model. Timer and K-M plotters were employed to find the expression and prognosis of SLC24A2 in pan cancer. Results: Interestingly, both DEGs between T2D verse control and SLC24A2-high verse SLC24A2-low enriched in cancer-related pathways. Moreover, a total of 3719 overlapped DEGs were divided into 8 functional modules. Grey module negatively correlated with T2D and FPG and was markedly involved in ribosome biogenesis. Ten SLC24A2-related genes (RRP36, RPF1, GRWD1, FBL, EXOSC5, BCCIP, UTP14A, TWISTNB, TBL3, and SKIV2L) were identified as hub genes, based on which the LASSO model accurately predicts the occurrence of T2D (AUC = 0.841). In addition, SLC24A2 was only expressed in islet ß cells and showed abnormal expression in 17 kinds of cancers and significantly correlated with the prognosis of 10 kinds of cancers. Conclusion: Taken together, SLC24A2 may link T2D and cancer by influencing the ribosome function of islet ß cells and play different prognostic roles in different cancers.