Molecular and Cellular Biology最新文献

RNASE1 is a ribonuclease secreted by cells and degrades extracellular RNAs. Here, we unexpectedly found that RNASE1, in addition to being secreted, is predominantly localized to the nucleus and functions to inhibit gene expression in human colorectal cancer (CRC) cells. RNASE1 expression is highly cell type-specific and is restricted to well-differentiated CRC cells where its transcription is activated by the pioneer transcription factor FOXA1. Using CRISPR interference utilizing three independent sgRNAs targeting the RNASE1 locus followed by RNA-seq, we found that upon depletion of RNASE1, most of the differentially expressed RNAs are modestly but significantly upregulated suggesting that RNASE1 predominantly functions to inhibit gene expression. In CRC patients, RNASE1 is significantly downregulated and high RNASE1 expression is associated with better patient survival, indicating a potential tumor suppressive function. Consistent with this, RNASE1 depletion results in increased proliferation and clonogenicity indicating that RNASE1 inhibits the growth of CRC cells. Finally, a promising RNASE1 target among the most significantly upregulated mRNAs upon RNASE1 depletion is DKK1 (Dickkopf inhibitor 1) which is upregulated in CRC and negatively regulated by RNASE1. Collectively, this initial characterization of endogenous RNASE1 uncovers a function of RNASE1 in inhibition of gene expression and CRC cell proliferation.

RNA modifications are highly conserved across all domains of life, suggesting an early emergence and a fundamental role in cellular processes. The modification 3-(3-amino-3-carboxypropyl)uridine (acp³U) is found in tRNAs of eukaryotes and prokaryotes, and in the 16S rRNA of archaea. In eukaryotic rRNA, a complex modification containing the acp group, m¹acp³Ψ is present at the analogous position. Although this modification was first identified in tRNA in 1969, only recently have the enzymes responsible for the synthesis of this modification on tRNA been identified. Despite its deep evolutionary conservation, the biological role of acp³U on tRNAs remains elusive. In Escherichia coli, it may contribute to genomic stability, while in human cells, loss of both tRNA acp³U-modifying enzymes impairs cell growth, though the underlying mechanisms are not yet understood. The conservation and multifunctionality of acp³U highlight the broader challenges of elucidating the roles of tRNA modifications in cellular homeostasis.

Over half of diabetes mellitus (DM) patients suffer from gastrointestinal motility disorders. miR-365-3p is involved in DM progression, but its role in gastrointestinal motility disorders remains unclear. This study explored whether miR-365-3p affects gastrointestinal motility in diabetic rats via the TLR4/MyD88/NF-κB pathway. A DM rat model was established using a high-fat, high-sugar diet and injected with a miR-365-3p mimic/inhibitor. DM symptoms, gastric emptying, intestinal propulsion rates, and gastrointestinal transit time were assessed. HE and TUNEL staining evaluated gastrointestinal pathology and apoptosis. qRT-PCR detected miR-365-3p levels, while ELISA assessed gastrointestinal motility-related factors. Immunofluorescence and Western blot analyzed C-kit, TLR4, and pathway proteins. DM rats exhibited increased body weight, blood glucose, and glucose intolerance, with reduced fasting insulin, confirming successful modeling. miR-365-3p was downregulated in DM rats. Injection of miR-365-3p mimic alleviated DM symptoms, reduced gastrointestinal tissue damage and apoptosis, and improved motility. The TLR4 agonist CRX-527 impaired these effects. In conclusion, miR-365-3p overexpression alleviates DM symptoms, gastrointestinal injury, and motility disorders by inhibiting the TLR4/MyD88/NF-κB pathway, offering a potential therapeutic target.

In esophageal squamous cell carcinoma, genetic activation of NRF2 increases resistance to chemotherapy and radiotherapy, which results in a significantly worse prognosis for patients. Therefore NRF2-activated cancers create an urgent clinical need to identify new therapeutic options. In this context, we previously identified the geldanamycin family of HSP90 inhibitors, which includes 17DMAG, to be synthetic lethal with NRF2 activity. As the first-generation of geldanamycin-derivative drugs were withdrawn from clinical trials due to hepatotoxicity, we designed second-generation compounds with C19-substituted structures in order to inhibit glutathione conjugation-mediated hepatotoxicity. In this study, using a variety of in vitro and in vivo cancer models, we found that C19-substituted 17DMAG compounds maintain their enhanced toxicity profile and synthetic lethal interaction with NRF2-NQO1-activated cancer cells. Importantly, using a xenograft mouse tumor model, we found that C19-substituted 17DMAG displayed significant anticancer efficacy against NRF2-NQO1-activated cancer cells without causing hepatotoxicity. These results clearly demonstrate the improved clinical potential for this new class of HSP90 inhibitor anticancer drugs, and suggest that patients with NRF2-NQO1-activated esophageal carcinoma may benefit from this novel therapeutic approach.

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by cognitive and language impairments, seizures, reduced or fragmented sleep, motor ataxia, and a characteristic happy affect. AS arises due to the neuronal loss of UBE3A, an E3 ligase that regulates protein abundance through the addition of lysine 48 (K48)-linked polyubiquitin chains to proteins targeted for degradation by the ubiquitin proteasome system (UPS). Using a dual SMAD inhibition protocol to derive cortical neurons from human induced pluripotent stem cells, we examined UBE3A deletion effects on the neuronal proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS identified 645 proteins differentially abundant between UBE3A knockout (KO) and isogenic UBE3A wild-type control cortical neurons. Proteins with increased abundance with UBE3A loss of function include GRIPAP1 and PACSIN1, synaptic proteins implicated in AMPA receptor recycling. We provide evidence UBE3A polyubiquitinates PACSIN1 and GRIPAP1 to regulate protein turnover, with potential implications for impaired activity-dependent synaptic plasticity observed in AS.

Chromatin regulators are frequently mutated in autism spectrum disorders, but in most cases how they cause disease is unclear. Mutations in the activity dependent neuroprotective protein (ADNP) causes ADNP syndrome, which is characterized by intellectual deficiency and developmental delays. To identify mechanisms that contribute to ADNP syndrome, we used induced pluripotent stem cells derived from ADNP syndrome patients as a model to test the effects of syndromic ADNP mutations on gene expression and neurodifferentiation. We found that some ADNP mutations result in truncated ADNP proteins, which displayed aberrant subcellular localization. Gene expression analyses revealed widespread transcriptional deregulation in all tested mutants. Interestingly, mutants that show presence of ADNP fragments show ER stress as evidenced by activation of the unfolded protein response (UPR). The mutants showing the greatest UPR pathway activation associated with the most severe neurodifferentiation and survival defects. Our results reveal the potential to explore UPR activation as a new biomarker for ADNP syndrome severity and perhaps also in other ASDs where mutations result in presence of truncated proteins.

Chromatin structure in eukaryotes is organized into functional domains through protein-DNA complexes. The cis-acting DNA elements are attached to the nuclear matrix, known as scaffold/matrix attachment regions (S/MARs). The associated protein partners known as matrix-associated region binding proteins (MARBPs). The coordinated interactions between MARBP and MARs to the nuclear scaffold act as an anchor for chromatin attachment and influences the regulation of gene expression. MARBPs modulate local epigenetic landscape of chromatin through the epigenetic modifiers. This function is executed by participating in the posttranslational modifications (PTMs) of DNA and histones. Such epigenetic changes are governed by crosstalk between long noncoding RNAs (lncRNAs) and associated MARBPs. Thus, dysregulation of either MARBPs or lncRNAs may alter gene expression potentially contributing to various disease manifestations. In this review, we elaborate on regulatory crosstalk between lncRNAs and MARBPs, its implication in human diseases, and possible therapeutics.

Midnolin (Midn) was originally discovered as a gene expressed specifically in the mouse midbrain at the embryonic developmental stage; MIDN was localized in the nucleus/nucleolus. Although the pathophysiological roles of MIDN remained largely unknown for many years after its discovery, its molecular functions and relevance to diseases have gradually become clearer. In PC12 cells, a rat neuronal model cell line, liquidity factors that are necessary for neurite outgrowth are reported to induce Midn gene expression. In addition, MIDN is required for E3 ubiquitin-protein ligase parkin expression, suggesting that MIDN is important for the development and maintenance of neuronal functions. Notably, it was recently reported that MIDN plays fundamental roles in the ubiquitin-independent proteasomal degradation of various nuclear proteins and transcription factors. Regarding the relationship between MIDN and diseases, copy number loss of MIDN is associated with Parkinson's disease, suggesting that MIDN is a genetic risk factor for this disease. In addition, MIDN is relevant to many types of malignant cancer, including B-cell lymphoma and liver cancer. Thus, MIDN is an essential molecule for the maintenance of homeostasis, and its functional disorder triggers multiple diseases depending on the affected tissues/organs. MIDN therefore shows promise as a potential therapeutic target and prognostic biomarker.

Previous structural and biochemical studies revealed that a negatively charged intrinsically disordered region (IDR) at the C-terminal of the Spt16 subunit of an evolutionarily conserved heterodimeric histone chaperone, FACT (Facilitates chromatin transcription), interacts with histone H2A-H2B dimer, and hence interferes the interaction of DNA with histone H2A-H2B dimer. However, the functional relevance of the binding of Spt16's IDR to histone H2A-H2B dimer with impact on chromatin dynamics and transcription has not been clearly elucidated in living cells. Here, we show that Spt16's IDR facilitates the eviction of histone H2A-H2B dimer (and hence chromatin disassembly) from the inducible GAL promoters upon transcription induction. Such facilitation of chromatin disassembly by Spt16's IDR stimulates the pre-initiation complex (PIC) formation at the promoter, and hence transcription initiation. Further, we find that Spt16's IDR regulates chromatin reassembly at the coding sequence in the wake of elongating RNA polymerase II. Collectively, our results reveal that Spt16's IDR facilitates promoter chromatin disassembly for stimulation of the PIC formation for transcription initiation with additional function in chromatin reassembly at the coding sequence in the wake of elongating RNA polymerase II, thus illuminating novel IDR regulation of chromatin dynamics and transcription in vivo.

The cytoplasmic tyrosine kinase Src supports many phenotypes in cancer cells, including proliferation, migration and invasion, survival, and metastasis. We have previously shown that Src promotes cytoplasmic localization of the RhoGEF Net1, where it stimulates RhoA activation, breast cancer cell motility, and extracellular matrix invasion. In the present work, we show that the Net1 expression in human breast tumors correlates with Src phosphorylation on its activating site Y419. We also show in human breast cancer cell lines that endogenous Net1 and Src interact, and that Net1 expression is required for full Src activation. Net1 must localize to the cytosol to promote Src activation, but surprisingly, the catalytic activity of Net1 toward Rho GTPases is not necessary for Src activation. Instead, Net1 requires interaction with the scaffolding protein Dlg1. Dlg1 knockdown prevents Src activation by Net1 and precludes interaction between Net1 and Src. Moreover, Net1 knockdown cooperates with small molecule inhibition of Src to inhibit breast cancer cell motility and extracellular matrix invasion. These data show a previously unrecognized relationship between Net1 and Src in human breast tumors and breast cancer cell lines, and suggest that therapeutic targeting of Net1 may be of benefit in breast cancers with elevated Src activity.