Exploring the brain epitranscriptome: perspectives from the NSAS summit.

Increasing evidence reinforces the essential function of RNA modifications in development and diseases, especially in the nervous system. RNA modifications impact various processes in the brain, including neurodevelopment, neurogenesis, neuroplasticity, learning and memory, neural regeneration, neurodegeneration, and brain tumorigenesis, leading to the emergence of a new field termed neuroepitranscriptomics. Deficiency in machineries modulating RNA modifications has been implicated in a range of brain disorders from microcephaly, intellectual disability, seizures, and psychiatric disorders to brain cancers such as glioblastoma. The inaugural NSAS Challenge Workshop on Brain Epitranscriptomics hosted in Crans-Montana, Switzerland in 2023 assembled a group of experts from the field, to discuss the current state of the field and provide novel translational perspectives. A summary of the discussions at the workshop is presented here to simulate broader engagement from the general neuroscience field.

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance.

FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.

tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation.

tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. Here we describe a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, we used small RNAs Illumina sequencing datasets extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (dTrm7_34 and dTrm7_32). Using our pipeline, we show how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology.

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

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.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

Isolation of small interfering RNAs using viral suppressors of RNA interference.

The tombusvirus P19 VSR (viral suppressor of RNA interference) binds siRNAs with high affinity, whereas the Flockhouse Virus (FHV) B2 VSR binds both long double-stranded RNA (dsRNA) and small interfering RNAs (siRNAs). Both VSRs are small proteins and function in plant and animal cells. Fusing a Nuclear Localization Signal (NLS) to the N-terminus shifts the localization of the VSR from cytoplasmic to nuclear, allowing researchers to specifically probe the subcellular distribution of siRNAs, and to investigate the function of nuclear and cytoplasmic siRNAs. This chapter provides a detailed protocol for the immunoprecipitation of siRNAs bound to epitope-tagged VSR and subsequent analysis by 3'-end-labeling using cytidine-3',5'-bis phosphate ([5'-(32)P]pCp) and northern blotting.

The Drosophila NURF remodelling and the ATAC histone acetylase complexes functionally interact and are required for global chromosome organization.

Drosophila Gcn5 is the catalytic subunit of the SAGA and ATAC histone acetylase complexes. Here, we show that mutations in Gcn5 and the ATAC component Ada2a induce a decondensation of the male X chromosome, similar to that induced by mutations in the Iswi and Nurf301 subunits of the NURF nucleosome remodelling complex. Genetic studies as well as transcript profiling analysis indicate that ATAC and NURF regulate overlapping sets of target genes during development. In addition, we find that Ada2a chromosome binding and histone H4-Lys12 acetylation are compromised in Iswi and Nurf301 mutants. Our results strongly suggest that NURF is required for ATAC to access the chromatin and to regulate global chromosome organization.