Functional interplay within the epitranscriptome: Reality or fiction?

RNA modifications have recently emerged as an important regulatory layer of gene expression. The most prevalent and reversible modification on messenger RNA (mRNA), N6-methyladenosine, regulates most steps of RNA metabolism and its dysregulation has been associated with numerous diseases. Other modifications such as 5-methylcytosine and N1-methyladenosine have also been detected on mRNA but their abundance is lower and still debated. Adenosine to inosine RNA editing is widespread on coding and non-coding RNA and can alter mRNA decoding as well as protect against autoimmune diseases. 2'-O-methylation of the ribose and pseudouridine are widespread on ribosomal and transfer RNA and contribute to proper RNA folding and stability. While the understanding of the individual role of RNA modifications has now reached an unprecedented stage, still little is known about their interplay in the control of gene expression. In this review we discuss the examples where such interplay has been observed and speculate that with the progress of mapping technologies more of those will rapidly accumulate

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila

Contains fulltext : 230120.pdf (publisher's version ) (Open Access)29 januari 202

Biallelic variants in NSUN6 cause an autosomal recessive neurodevelopmental disorder.

5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive. We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene. We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment. Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition

Mechanisms of targeting the MDM2-p53-FOXM1 axis in well-differentiated intestinal neuroendocrine tumors

BACKGROUND/AIMS: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of p53 negative regulators, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo. METHODS: By western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immune fluorescence, western blot and multiplex gene expression analysis. Finally, we analyzed the anti-tumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. RESULTS: The midgut wild type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins re-activated an anti-proliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decrease tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced anti-proliferative effects. CONCLUSION: In summary, MDM2 overexpression is a frequent event in p53 wild type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m <sup>6</sup> A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m <sup>6</sup> A alters fly behavior, albeit the underlying molecular mechanism and the role of m <sup>6</sup> A during nervous system development have remained elusive. Here we find that impairment of the m <sup>6</sup> A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m <sup>6</sup> A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m <sup>6</sup> A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m <sup>6</sup> A pathway controls development of the nervous system and modulates Fmr1 target transcript selection

Supplementary Material for: Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors

<p><b><i>Background/Aims:</i></b> The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo<i>.</i><b><i>Methods:</i></b> By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. <b><i>Results:</i></b> The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. <b><i>Conclusion:</i></b> In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.</p