Mass Spectrometry-Based Pipeline for Identifying RNA Modifications Involved in a Functional Process: Application to Cancer Cell Adaptation.

Cancer onset and progression are known to be regulated by genetic and epigenetic events, including RNA modifications (a.k.a. epitranscriptomics). So far, more than 150 chemical modifications have been described in all RNA subtypes, including messenger, ribosomal, and transfer RNAs. RNA modifications and their regulators are known to be implicated in all steps of post-transcriptional regulation. The dysregulation of this complex yet delicate balance can contribute to disease evolution, particularly in the context of carcinogenesis, where cells are subjected to various stresses. We sought to discover RNA modifications involved in cancer cell adaptation to inhospitable environments, a peculiar feature of cancer stem cells (CSCs). We were particularly interested in the RNA marks that help the adaptation of cancer cells to suspension culture, which is often used as a surrogate to evaluate the tumorigenic potential. For this purpose, we designed an experimental pipeline consisting of four steps: (1) cell culture in different growth conditions to favor CSC survival; (2) simultaneous RNA subtype (mRNA, rRNA, tRNA) enrichment and RNA hydrolysis; (3) the multiplex analysis of nucleosides by LC-MS/MS followed by statistical/bioinformatic analysis; and (4) the functional validation of identified RNA marks. This study demonstrates that the RNA modification landscape evolves along with the cancer cell phenotype under growth constraints. Remarkably, we discovered a short epitranscriptomic signature, conserved across colorectal cancer cell lines and associated with enrichment in CSCs. Functional tests confirmed the importance of selected marks in the process of adaptation to suspension culture, confirming the validity of our approach and opening up interesting prospects in the field.

Multiplexed LC-MS/MS quantification of salivary RNA modifications in periodontitis.

OBJECTIVE: To analyse the salivary epitranscriptomic profiles as periodontitis biomarkers using multiplexed mass spectrometry (MS). BACKGROUND: The field of epitranscriptomics, which relates to RNA chemical modifications, opens new perspectives in the discovery of diagnostic biomarkers, especially in periodontitis. Recently, the modified ribonucleoside N6-methyladenosine (m6A) was revealed as a crucial player in the etiopathogenesis of periodontitis. However, no epitranscriptomic biomarker has been identified in saliva to date. MATERIALS AND METHODS: Twenty-four saliva samples were collected from periodontitis patients (n = 16) and from control subjects (n = 8). Periodontitis patients were stratified according to stage and grade. Salivary nucleosides were directly extracted and, in parallel, salivary RNA was digested into its constituent nucleosides. Nucleoside samples were then quantified by multiplexed MS. RESULTS: Twenty-seven free nucleosides were detected and an overlapping set of 12 nucleotides were detected in digested RNA. Among the free nucleosides, cytidine and three other modified nucleosides (inosine, queuosine and m6Am) were significantly altered in periodontitis patients. In digested RNA, only uridine was significantly higher in periodontitis patients. Importantly there was no correlation between free salivary nucleoside levels and the levels of those same nucleotides in digested salivary RNA, except for cytidine, m5C and uridine. This statement implies that the two detection methods are complementary. CONCLUSION: The high specificity and sensitivity of MS allowed the detection and quantification of multiple nucleosides from RNA and free nucleosides in saliva. Some ribonucleosides appear to be promising biomarkers of periodontitis. Our analytic pipeline opens new perspectives for diagnostic periodontitis biomarkers.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology.

Despite significant progress in targeted therapies, cancer recurrence remains a major cause of mortality worldwide. Identification of accurate biomarkers, through molecular profiling in healthy and cancer patient samples, will improve diagnosis and promote personalized medicine. While genetic and epigenetic alterations of DNA are currently exploited as cancer biomarkers, their robustness is limited by tumor heterogeneity. Recently, cancer-associated changes in RNA marks have emerged as a promising source of diagnostic and prognostic biomarkers. RNA epigenetics (also known as epitranscriptomics) is an emerging field in which at least 150 chemical modifications in all types of RNA (mRNA, tRNA, lncRNA, rRNA, and microRNA) have been detected. These modifications fine-tune gene expression in both physiological and pathological processes. A growing number of studies have established links between specific modified nucleoside levels in solid/liquid biopsies, and cancer onset and progression. In this review, we highlight the potential role of epitranscriptomic markers in refining cancer diagnosis and/or prognosis. RNA modification patterns may contain important information for establishing an initial diagnosis, monitoring disease evolution, and predicting response to treatment. Furthermore, recent developments in mass spectrometry allow reliable quantification of RNA marks in solid biopsies and biological fluids. We discuss the great potential of mass spectrometry for identifying epitranscriptomic biomarker signatures in cancer diagnosis. While there are various methods to quantify modified nucleosides, most are unable to detect and quantify more than one type of RNA modification at a time. Mass spectrometry analyses, especially GC-MS/MS and LC-MS/MS, overcome this limitation and simultaneously detect modified nucleosides by multiple reaction monitoring. Indeed, several groups are currently validating mass spectrometry methods that quantify several nucleosides at one time in liquid biopsies. The challenge now is to exploit these powerful analytical tools to establish epitranscriptomic signatures that should open new perspectives in personalized medicine. This review summarizes the growing clinical field of analysis of RNA modifications and discusses pre-analytical and analytical approaches, focusing in particular on the development of new mass spectrometry tools and their clinical applications.

FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell.

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.

The m6A pathway protects the transcriptome integrity by restricting RNA chimera formation in plants.

Global, segmental, and gene duplication-related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)-assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3'-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.