Post-transcriptional methylation of N6- and N1-adenine can effect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA lead to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.

Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to sitespecifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art singlemolecule experiments.

At any stage of their lifecycle, mRNAs are coated by specialized proteins. One of few circumstances when free mRNA appears in the cytosol is the disassembly of polysomes during the stress-induced shutdown of protein synthesis. Using quantitative mass spectrometry, we sought to identify the free RNA-interacting cellular machinery in heat-shocked mammalian cells. Free RNA-associated proteins displayed higher disorder and larger size, which supports the role of multivalent interactions during the initial phase of the association with RNAs during stress. Structural features of the free RNA interactors defined them as a subset of RNA-binding proteins. The interaction between these assembled proteins in vivo required RNA. Reconstitution of the association process in vitro indicated a multimolecular basis for increased binding to RNA upon heat shock in the cytosol. Our study represents a step toward understanding how free RNA is processed in the cytosol during proteostasis stress.

H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.

H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.