ChemRAP uncovers specific mRNA translation regulation via RNA 5' phospho-methylation.

5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.

BCDIN3D regulates tRNAHis 3' fragment processing.

5' ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5' monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3' end of tRNAHis. Moreover, we were able to generate this 'miRNA' in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5'P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3'-fragment/'miR-4454' levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3'-fragment processing without negatively affecting tRNAHis's canonical function of aminoacylation.

Deletion of the neural tube defect-associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos.

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.

BCDIN3D RNA methyltransferase stimulates Aldolase C expression and glycolysis through let-7 microRNA in breast cancer cells.

Type II diabetes (T2D) and specific cancers share many risk factors, however, the molecular mechanisms underlying these connections are often not well-understood. BCDIN3D is an RNA modifying enzyme that methylates specific precursor microRNAs and tRNAHis. In addition to breast cancer, BCDIN3D may also be linked to metabolism, as its gene locus is associated with obesity and T2D. In order to uncover metabolic pathways regulated by BCDIN3D in cancer, we performed an unbiased analysis of the metabolome, transcriptome, and proteome of breast cancer cells depleted for BCDIN3D. Intersection of these analyses showed that BCDIN3D-depleted cells have increased levels of Fructose 1,6 Bisphosphate (F1,6-BP), the last six-carbon glycolytic intermediate accompanied by reduced glycolytic capacity. We further show that elevated F1,6-BP is due to downregulation of Aldolase C (ALDOC), an enzyme that cleaves F1,6-BP mainly in the brain, but whose high expression/amplification is associated with poor prognosis in breast cancer. BCDIN3D regulates ALDOC through a non-canonical mechanism involving the crucial let-7 microRNA family and its target site on the 3'UTR of ALDOC. Overall, our results reveal an important connection between BCDIN3D, let-7 and glycolysis that may be relevant to breast cancer, obesity, and T2D.

Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine.

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.

The Yin and Yang of Microrna Assay Methods.

OBJECTIVE: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. METHODS: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to AffymetrixTM arrays, qPCR and digital droplet qPCR (BioradTM) based on Taqman microRNA assays (Life TechnologiesTM). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. RESULTS: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. CONCLUSION: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

The Yin and Yang of microRNA Assay Methods.

OBJECTIVE: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. METHODS: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to AffymetrixTM arrays, qPCR and digital droplet qPCR (BioradTM) based on Taqman microRNA assays (Life TechnologiesTM). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. RESULTS: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. CONCLUSION: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

Exosomal MicroRNAs in Tumoral U87 MG Versus Normal Astrocyte Cells.

Brain glial tumors, and particularly glioblastomas, are tumors with a very poor prognosis. Currently, the parameters that control aggressiveness, migration, or chemoresistance are not well known. In this tumor context, microRNAs are thought to be essential actors of tumorigenesis as they are able to control the expression of numerous genes. microRNAs are not only active in controlling tumor cell pathways, they are also secreted by cells, inside microvesicles called exosomes, and may play specific roles outside the tumor cells in the tumor microenvironment. We analyzed the microRNA content of exosomes produced in vitro by normal glial cells (astrocytes) and tumor glial cells (U87 MG) using Affymetrix microarrays. It appears that the exosome microRNA profiles are qualitatively quite similar. Nevertheless, their quantitative profiles are different and may be potentially taken as an opportunity to carry out assays of diagnostic interest. We submitted the cultured cells to several stresses such as oxygen deprivation or treatments with chemical drugs (GW4869 or 5-Aza-2'- deoxycitidine) to assess the impact of the cellular microRNA profile modifications on the exosome microRNA profiles. We found that modifications of the cellular microRNA content are not strictly mirrored in exosomes. On the basis of these results, we propose that the way microRNAs are released in exosomes is probably the result of a combination of different excretion mechanisms or constraints that concur in a controlled regulation of the exosome microRNA secretion.

MicroRNAs: molecular features and role in cancer.

microRNAs (miRNAs) are small noncoding endogenously produced RNAs that play key roles in controlling the expression of many cellular proteins. Once they are recruited and incorporated into a ribonucleoprotein complex miRISC, they can target specific mRNAs in a miRNA sequence-dependent process and interfere in the translation into proteins of the targeted mRNAs via several mechanisms. Consequently, miRNAs can regulate many cellular pathways and processes. Dysregulation of their physiological roles may largely contribute to disease. In particular, in cancer, miRNAs can be involved in the deregulation of the expression of important genes that play key roles in tumorigenesis, tumor development, and angiogenesis and have oncogenic or tumor suppressor roles. This review focuses on the biogenesis and maturation of miRNAs, their mechanisms of gene regulation, and the way their expression is deregulated in cancer. The involvement of miRNAs in several oncogenic pathways such as angiogenesis and apoptosis, and in the inter-cellular dialog mediated by miRNA-loaded exosomes as well as the development of new therapeutical strategies based on miRNAs will be discussed.

MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes.

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.