The N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine ...RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1). We demonstrate that WEE1 transcripts contain m6A modification, which promotes their decay through YTHDF2. Moreover, we found that YTHDF2 protein stability is dependent on cyclin-dependent kinase 1 (CDK1) activity. Thus, CDK1, YTHDF2, and WEE1 form a feedforward regulatory loop to promote mitotic entry. We further identified Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1 (DDB1), and S-phase kinase-associated protein 2 (SKP2) as components of E3 ubiquitin ligase complexes that mediate YTHDF2 proteolysis. Our study provides insights into how cell cycle mediators modulate transcriptomic m6A modification, which in turn regulates the cell cycle.
Targeted inhibition of oncogenic miRNA-21 has been proposed to treat glioblastoma by rescuing tumor suppressors, PTEN and PDCD4. However, systemic delivery of anti-miR-21 sequences requires a robust ...and efficient delivery platform to successfully inhibit this druggable target. Three-way-junction (3WJ)-based RNA nanoparticles (RNP), artificially derived from pRNA of bacteriophage phi29 DNA packaging motor, was recently shown to target glioblastoma. Here, we report that multi-valent folate (FA)-conjugated 3WJ RNP constructed to harbor anti-miR-21 LNA sequences (FA-3WJ-LNA-miR21) specifically targeted and delivered anti-miR-21 LNA and knocked down miR-21 expression in glioblastoma cells in vitro and in vivo with favorable biodistribution. Systemically injected FA-3WJ-LNA-miR21 RNP efficiently rescued PTEN and PDCD4, resulting in glioblastoma cell apoptosis and tumor growth regression. Overall survival rate was also significantly improved by FA-3WJ-LNA-miR21 RNP. These results are indicative of the clinical benefit of FA-3WJ RNP-based gene therapy for the successful targeted therapy of developing and even recurring glioblastoma.
Lee et al. show that RNA nanoparticle (RNP) configured based on three-way-junction (3WJ) of pRNA of bacteriophage phi29 successfully delivered anti-miR-21 LNA into glioblastoma cells and intracranial mouse brain glioblastoma and reduced tumor growth with significantly improved survival rate by rescuing tumor suppressor genes, demonstrating the therapeutic potential of 3WJ-based RNP as miRNA delivery platform for glioblastoma targeted therapy.
Link Between m6A Modification and Cancers Liu, Zhen-Xian; Li, Li-Man; Sun, Hui-Lung ...
Frontiers in bioengineering and biotechnology,
07/2018, Letnik:
6
Journal Article
Recenzirano
Odprti dostop
N6-methyladenosine (m6A) epitranscriptional modification has recently gained much attention. Through the development of m6A sequencing, the molecular mechanism and importance of m6A have been ...revealed. m6A is the most abundant internal modification in higher eukaryotic mRNAs, which plays crucial roles in mRNA metabolism and multiple biological processes. In this review, we introduce the characteristics of m6A regulators, including "writers" that create m6A mark, "erasers" that show demethylation activity and "readers" that decode m6A modification to govern the fate of modified transcripts. Moreover, we highlight the roles of m6A modification in several common cancers, including solid and non-solid tumors. The regulators of m6A exert enormous functions in cancer development, such as proliferation, migration and invasion. Especially, with the underlying mechanisms being uncovered, m6A and its regulators are expected to be the targets for the diagnosis and treatment of cancers.
20‐(S)‐Camptothecin (CPT)‐conjugated dipeptides are reported that preassemble into nanotubes with diameters ranging from 80–120 nm. These nanoassemblies maintain a high (∼47 %) drug loading and ...exhibit greater drug stability (i.e., resistance to lactone hydrolysis), and consequently greater efficacy against several human cancer cells (HT‐29, A549, H460, and H23) in vitro compared with the clinically used prodrug irinotecan. A key and defining feature of this system is the use of the CPT‐conjugated dipeptide as both the drug and precursor to the nanostructured carrier, which simplifies the overall fabrication process.
Happy campers: Camptothecin (CPT)‐conjugated dipeptides self‐assemble into nanotube delivery vehicles with high drug loading levels. These nanostructures display greater hydrolytic stability and greater efficacy against several human cancer cells (HT29, A549, H460, and H23) in vitro compared with the clinically used prodrug irinotecan.
5‐Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt‐tRNAMet) and cytosolic leucine tRNA (ct‐tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has ...also been detected in yeast mRNA and human tRNA, but its transcriptome‐wide distribution in mammals has not been studied. Here we report f5C‐seq, a quantitative sequencing method to map f5C transcriptome‐wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pic‐borane, and DHU can be exclusively read as T during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C‐to‐T mutation signature. We validated f5C‐seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1‐depleted cells. By applying f5C‐seq to chromatin‐associated RNA (caRNA), we identified several highly modified f5C sites in HeLa and mouse embryonic stem cells (mESC).
Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion ...sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.
The self-assembly of 5-fluorouracil dilysine conjugates into self-supporting hydrogels, comprised of entangled nanofibers or rigid nanotubes with diameters of 10 and 16 nm, respectively, is reported. ...The rate of release of 5-Fu from the conjugates was highly dependent on concentration in solution, whereas, release from the fully formed hydrogels was significantly slower. The 5-Fu conjugate also exhibited promising in vitro cytotoxicity against human tumor cell lines A549, H460 and H23.
Lung cancer is the leading cause of cancer-related deaths worldwide. Despite advancements and improvements in surgical and medical treatments, the survival rate of lung cancer patients remains ...frustratingly poor. Local control for early-stage nonsmall cell lung cancer (NSCLC) has dramatically improved over the last decades for both operable and inoperable patients. However, the molecular mechanisms of NSCLC invasion leading to regional and distant disease spread remain poorly understood. Here, we identifymicroRNA-224(miR-224) to be significantly up-regulated in NSCLC tissues, particularly in resected NSCLC metastasis. IncreasedmiR-224expression promotes cell migration, invasion, and proliferation by directly targeting the tumor suppressors TNFα-induced protein 1 (TNFAIP1) and SMAD4. In concordance with in vitro studies, mouse xenograft studies validated thatmiR-224functions as a potent oncogenic miRNA in NSCLC in vivo. Moreover, we found promoter hypomethylation and activated ERK signaling to be involved in the regulation ofmiR-224expression in NSCLC. Up-regulatedmiR-224, thus, facilitates tumor progression by shifting the equilibrium of the partially antagonist functions of SMAD4 and TNFAIP1 toward enhanced invasion and growth in NSCLC. Our findings indicate that targetingmiR-224could be effective in the treatment of certain lung cancer patients.
MicroRNAs (miRNA) are mostly downregulated in cancer. However, the mechanism underlying this phenomenon and the precise consequence in tumorigenesis remain obscure. Here we show that ERK suppresses ...pre-miRNA export from the nucleus through phosphorylation of exportin-5 (XPO5) at T345/S416/S497. After phosphorylation by ERK, conformation of XPO5 is altered by prolyl isomerase Pin1, resulting in reduction of pre-miRNA loading. In liver cancer, the ERK-mediated XPO5 suppression reduces miR-122, increases microtubule dynamics, and results in tumor development and drug resistance. Analysis of clinical specimens further showed that XPO5 phosphorylation is associated with poor prognosis for liver cancer patients. Our study reveals a function of ERK in miRNA biogenesis and suggests that modulation of miRNA export has potential clinical implications.
•ERK phosphorylation followed by Pin1-mediated isomerization impairs XPO5 activity•Downregulation of miR-122 leads to taxol resistance through septin-9 and MARK4•XPO5 phosphorylation correlates with poor prognosis in HCC patients•Pin1 and MARK4 are potential targets for clinical intervention in liver cancer
Sun et al. find that ERK phosphorylates XPO5, which induces a Pin1-mediated conformational change that inhibits the ability of XPO5 to load and export pre-miRNA from the nucleus. Phosphorylation of XPO5 is associated with global miRNA downregulation and correlates with poor survival in hepatocellular carcinoma.
The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and ...methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.
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•Expression of RNA methyltransferases METTL3/14 correlates with survival in neuroblastoma•Inhibition of METTL3/14 decreases cell survival and reduces tumor growth in xenograft models•METTL3/14 inhibition regulates RNA stability by mediating site-specific m6A loss•METTL3/14 inhibition up-regulates differentiation-associated transcriptional networks
Pomaville et al. show that the catalytic activity of the METTL3/14 complex is critical for neuroblastoma growth. Pharmacological inhibition of METTL3/14 promoted neuroblastoma differentiation and suppressed tumor xenograft growth. Inhibitor treatment mediated m6A loss at sites specific for neuronal differentiation, increasing transcript stability and the expression of neuronal differentiation networks.
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