Florian Pichot

Bio: Florian Pichot is an academic researcher from University of Mainz. The author has contributed to research in topics: RNA & Transfer RNA. The author has an hindex of 6, co-authored 7 publications receiving 163 citations. Previous affiliations of Florian Pichot include Centre national de la recherche scientifique & University of Lorraine.

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

Abstract: Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation

Abstract: Analysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing‐based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′‐O‐methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally‐important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′‐O‐methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species.

Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

Abstract: A major trend in the epitranscriptomics field over the last 5 years has been the high-throughput analysis of RNA modifications by a combination of specific chemical treatment(s), followed by library preparation and deep sequencing. Multiple protocols have been described for several important RNA modifications, such as 5-methylcytosine (m5C), pseudouridine (ψ), 1-methyladenosine (m1A), and 2'-O-methylation (Nm). One commonly used method is the alkaline cleavage-based RiboMethSeq protocol, where positions of reads' 5'-ends are used to distinguish nucleotides protected by ribose methylation. This method was successfully applied to detect and quantify Nm residues in various RNA species such as rRNA, tRNA, and snRNA. Such applications require adaptation of the initially published protocol(s), both at the wet bench and in the bioinformatics analysis. In this manuscript, we describe the optimization of RiboMethSeq bioinformatics at the level of initial read treatment, alignment to the reference sequence, counting the 5'- and 3'- ends, and calculation of the RiboMethSeq scores, allowing precise detection and quantification of the Nm-related signal. These improvements introduced in the original pipeline permit a more accurate detection of Nm candidates and a more precise quantification of Nm level variations. Applications of the improved RiboMethSeq treatment pipeline for different cellular RNA types are discussed.

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination

Abstract: Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA

Abstract: Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

RNA 2′-O-Methylation (Nm) Modification in Human Diseases

Abstract: Nm (2′-O-methylation) is one of the most common modifications in the RNA world. It has the potential to influence the RNA molecules in multiple ways, such as structure, stability, and interactions, and to play a role in various cellular processes from epigenetic gene regulation, through translation to self versus non-self recognition. Yet, building scientific knowledge on the Nm matter has been hampered for a long time by the challenges in detecting and mapping this modification. Today, with the latest advancements in the area, more and more Nm sites are discovered on RNAs (tRNA, rRNA, mRNA, and small non-coding RNA) and linked to normal or pathological conditions. This review aims to synthesize the Nm-associated human diseases known to date and to tackle potential indirect links to some other biological defects.

Noncanonical Roles of tRNAs: tRNA Fragments and Beyond.

Abstract: As one of the most abundant and conserved RNA species, transfer RNAs (tRNAs) are well known for their role in reading the codons on messenger RNAs and translating them into proteins. In this review, we discuss the noncanonical functions of tRNAs. These include tRNAs as precursors to novel small RNA molecules derived from tRNAs, also called tRNA-derived fragments, that are abundant across species and have diverse functions in different biological processes, including regulating protein translation, Argonaute-dependent gene silencing, and more. Furthermore, the role of tRNAs in biosynthesis and other regulatory pathways, including nutrient sensing, splicing, transcription, retroelement regulation, immune response, and apoptosis, is reviewed. Genome organization and sequence variation of tRNA genes are also discussed in light of their noncanonical functions. Lastly, we discuss the recent applications of tRNAs in genome editing and microbiome sequencing.