m6A methylation of transcription leader sequence impacts discontinuous transcription of subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative populations in equilibrium, suggesting that m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase.

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development.

Reinvestigating the clinical relevance of the m6A writer METTL3 in urothelial carcinoma of the bladder.

METTL3 is the major writer of N6-Methyladenosine (m6A) and has been associated with controversial roles in cancer. This is best illustrated in urothelial carcinoma of the bladder (UCB), where METTL3 was described to have both oncogenic and tumor-suppressive functions. Here, we reinvestigated the role of METTL3 in UCB. METTL3 knockout reduced the oncogenic phenotype and m6A levels of UCB cell lines. However, complete depletion of METTL3/m6A was not achieved due to selection of cells expressing alternative METTL3 isoforms. Systematic vulnerability and inhibitor response analyses suggested that uroepithelial cells depend on METTL3 for viability. Furthermore, expression and survival analyses of clinical data revealed a complex role for METTL3 in UCB, with decreased m6A mRNA levels in UCB tumors. Our results suggest that METTL3 expression may be a suitable diagnostic UCB biomarker, as the enzyme promotes UCB formation. However, the suitability of the enzyme as a therapeutic target should be evaluated carefully.

HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs.

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics.

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA's secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.

General Principles for the Detection of Modified Nucleotides in RNA by Specific Reagents.

Epitranscriptomics heavily rely on chemical reagents for the detection, quantification, and localization of modified nucleotides in transcriptomes. Recent years have seen a surge in mapping methods that use innovative and rediscovered organic chemistry in high throughput approaches. While this has brought about a leap of progress in this young field, it has also become clear that the different chemistries feature variegated specificity and selectivity. The associated error rates, e.g., in terms of false positives and false negatives, are in large part inherent to the chemistry employed. This means that even assuming technically perfect execution, the interpretation of mapping results issuing from the application of such chemistries are limited by intrinsic features of chemical reactivity. An important but often ignored fact is that the huge stochiometric excess of unmodified over-modified nucleotides is not inert to any of the reagents employed. Consequently, any reaction aimed at chemical discrimination of modified versus unmodified nucleotides has optimal conditions for selectivity that are ultimately anchored in relative reaction rates, whose ratio imposes intrinsic limits to selectivity. Here chemical reactivities of canonical and modified ribonucleosides are revisited as a basis for an understanding of the limits of selectivity achievable with chemical methods.

Binding and/or hydrolysis of purine-based nucleotides is not required for IM30 ring formation.

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.