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

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila. We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-…

Absolute Quantifizierung nicht‐kodierender RNA‐Spezies mittels Mikroskala‐Thermophorese

Absolute quantification of noncoding RNA by microscale thermophoresis

Abstract Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA‐Seq‐based quantification approaches, strongly suggesting a bias due to tRNA modifica…

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 lar…

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

AbstractCytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associate…