dTrmt10A impacts Hsp70 chaperone m6A levels and the stress response in the Drosophila brain.

Chronic cellular stress has a profound impact on the brain, leading to degeneration and accelerated aging. Recent work has revealed the vital role of RNA modifications, and the proteins responsible for regulating them, in the stress response. In our study, we defined the role of CG14618/dTrmt10A, the Drosophila counterpart of human TRMT10A a N1-methylguanosine methyltransferase, on m6A regulation and heat stress resilience in the Drosophila brain. By m6A-IP RNA sequencing on Drosophila head tissue, we demonstrated that manipulating dTrmt10A levels indirectly regulates m6A levels on polyA + RNA. dTrmt10A exerted its influence on m6A levels on transcripts enriched for neuronal signaling and heat stress pathways, similar to the m6A methyltransferase Mettl3. Intriguingly, its impact primarily targeted 3' UTR m6A, setting it apart from the majority of Drosophila m6A-modified transcripts which display 5' UTR enrichment. Upregulation of dTrmt10A led to increased resilience to acute heat stress, decreased m6A modification on heat shock chaperones, and coincided with decreased decay of chaperone transcripts and increased translation of chaperone proteins. Overall, these findings establish a potential mechanism by which dTrmt10A regulates the acute brain stress response through m6A modification.

Mettl3-catalyzed m6A regulates histone modifier and modification expression in self-renewing somatic tissue.

N6-methyladenosine (m6A) is the most abundant modification on messenger RNAs (mRNAs) and is catalyzed by methyltransferase-like protein 3 (Mettl3). To understand the role of m6A in a self-renewing somatic tissue, we deleted Mettl3 in epidermal progenitors in vivo. Mice lacking Mettl3 demonstrate marked features of dysfunctional development and self-renewal, including a loss of hair follicle morphogenesis and impaired cell adhesion and polarity associated with oral ulcerations. We show that Mettl3 promotes the m6A-mediated degradation of mRNAs encoding critical histone modifying enzymes. Depletion of Mettl3 results in the loss of m6A on these mRNAs and increases their expression and associated modifications, resulting in widespread gene expression abnormalities that mirror the gross phenotypic abnormalities. Collectively, these results have identified an additional layer of gene regulation within epithelial tissues, revealing an essential role for m6A in the regulation of chromatin modifiers, and underscoring a critical role for Mettl3-catalyzed m6A in proper epithelial development and self-renewal.

Noncatalytic regulation of 18S rRNA methyltransferase DIMT1 in acute myeloid leukemia.

Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.

Oxazolidinone-Mediated Sequence Determination of One-Bead One-Compound Cyclic Peptide Libraries.

A novel one-bead one-compound (OBOC) dual ring-opening/cleavage approach for cyclic peptide sequencing was developed. The method selectively modifies serine, cysteine, threonine, and/or glutamic acid to an oxazolidinone-derived moiety, thereby increasing the susceptibility of the modified peptide backbone toward hydrolysis. The resulting linear peptide was then sequenced in 1 min by tandem mass spectrometry on a quadrupole time-of-flight instrument incorporating two-dimensional liquid chromatography and ion mobility spectrometry separation. To evaluate this approach, a library of cyclic peptides was successfully sequenced with 98% overall accuracy, demonstrating its robustness and broad substrate scope.

Cyclic and Lasso Peptides: Sequence Determination, Topology Analysis, and Rotaxane Formation.

A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide-based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin-1 ΔC5.