The combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet, the molecular determinants governing interactions in protein-RNA networks are not well understood. Here we investigated the relationship between the structure of an RNA and its ability to interact with proteins. Analysing in silico, in vitro and in vivo experiments, we find that the amount of double-stranded regions in an RNA correlates with the number of protein contacts. This relationship —which we call structure-driven protein interactivity— allows classification of RNA types, plays a role in gene regulation and could have implications for the formation of phase-separated ribonucleoprotein assemblies. We validate our hypothesis by showing that a highly structured RNA can rearrange the composition of a protein aggregate. We report that the tendency of proteins to phase-separate is reduced by interactions with specific RNAs.

Significance: Newly synthetized enzymes must fold correctly and associate with cofactors to reach their functional state. Mutations often interfere with the structural and functional maturation and lead to unstable proteins. Here we demonstrate that cofactor deprivation caused by vitamin B2 deficiency results in the destabilization of a significant fraction of flavin-containing enzymes, even if they have wild-type sequences. The accumulation of conditionally unstable proteins would overload cellular protein degradation systems; consequently, these proteins would contribute to amyloidogenesis and protein aggregation disorders. Because nutritional deficits often develop in aging organisms, this study provides an additional, protein stability-based explanation of the connection between vitamin supplies and healthy aging.

Post-transcriptional methylation of N6-adenine and N1-adenine can affect 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 leads 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.

Significance: The desmosome is a major cell–cell junction connecting cells in tissues under high mechanical load. Currently, while structures of the constituent cadherins are known, the desmosome architecture has remained elusive. The primary reason is the high plasticity of the cadherins. As many other cellular structures, their high flexibility cannot be easily addressed by conventional structural techniques that rely on averaging many identical structures. For this, we combine high-end cryo-electron tomography with large-scale molecular dynamics simulations to produce a molecular model of the desmosome that integrates new with decades-old observations, accounts for the remarkable biophysical properties, and maps the intermolecular interactions.

Tumor cells adapt their metabolism to meet the energetic and anabolic requirements of high proliferation and invasiveness. The metabolic addiction has motivated the development of therapies directed at individual biochemical nodes. However, currently there are few possibilities to target multiple enzymes in tumors simultaneously. Flavin-containing enzymes, ca. 100 proteins in humans, execute key biotransformations in mammalian cells. To expose metabolic addiction, we inactivated a substantial fraction of the flavoproteome in melanoma cells by restricting the supply of the FMN and FAD precursor riboflavin, the vitamin B2. Vitamin B2 deficiency affected stability of many polypeptides and thus resembled the chaperone HSP90 inhibition, the paradigmatic multiple-target approach. In support of this analogy, flavin-depleted proteins increasingly associated with a number of proteostasis network components, as identified by the mass spectrometry analysis of the FAD-free NQO1 aggregates. Proteome-wide analysis of the riboflavin-starved cells revealed a profound inactivation of the mevalonate pathway of cholesterol synthesis, which underlines the manifold cellular vulnerability created by the flavoproteome inactivation. Cell cycle-arrested tumor cells became highly sensitive to alkylating chemotherapy. Our data suggest that the flavoproteome is well suited to design synthetic lethality protocols combining proteostasis manipulation and metabolic reprogramming.

C-terminal polylysine (PL) can be synthesized from the polyadenine tail of prematurely cleaved mRNAs or when a read-though of a stop codon happens. Due to the highly positive charge, PL stalls in the electrostatically negative ribosomal exit channel. The stalled polypeptide recruits the Ribosome-associated quality control (RQC) complex which processes and extracts the nascent chain. Dysfunction of the RQC leads to the accumulation of PL-tagged proteins, induction of stress response and cellular toxicity. Not much has been known about the PL-specific aspect of protein quality control. Using quantitative mass spectrometry, we uncovered the post-ribosomal PL-processing machinery in the human cytosol. It encompasses key cytosolic complexes of the proteostasis network, such as chaperonin TRiC and half-capped 19S-20S proteasome. Furthermore, we found that the nuclear transport machinery associates with PL which suggests a novel mechanism to compartmentalize faulty proteins in the cell. The enhanced nuclear import of a PL-tagged polypeptide confirmed this implication which raises the question regarding the biological rationale behind it.