RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33 C and 37 C) and reduced at room temperature (22 C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

Background:The mechanisms by which human PUF proteins repress target mRNAs remain unknown. Results: PUM1 and PUM2 reduce protein and mRNA levels of targets by recruiting the CNOT deadenylase complex and by a poly(A)-independent mechanism. Conclusion: PUMs employ deadenylation-dependent and -independent mechanisms of repression. Significance: Deadenylation is a conserved means of PUF repression but additional mechanism(s) contribute to mRNA regulation.

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.DOI: http://dx.doi.org/10.7554/eLife.17096.001

Drosophila melanogasterPumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.

PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3 ′ untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation.

Chemical probing is an important tool for characterizing the complex folded structures of RNA molecules, many of which play key cellular roles. Electrophilic SHAPE reagents create adducts at the 2′-hydroxyl position on the RNA backbone of flexible ribonucleotides with relatively little dependence on nucleotide identity. Strategies for adduct detection such as mutational profiling (MaP) allow accurate, automated calculation of relative adduct frequencies for each nucleotide in a given RNA or group of RNAs. A number of alternative reagents and adduct detection strategies have been proposed, especially for use in living cells. Here we evaluate five SHAPE reagents: three previously well-validated reagents 1M7 (1-methyl-7-nitroisatoic anhydride), 1M6 (1-methyl-6-nitroisatoic anhydride), and NMIA (N-methylisatoic anhydride), one more recently proposed NAI (2-methylnicotinic acid imidazolide), and one novel reagent 5NIA (5-nitroisatoic anhydride). We clarify the importance of carefully designed software in reading out SHAPE experiments using massively parallel sequencing approaches. We examine SHAPE modification in living cells in diverse cell lines, compare MaP and reverse transcription− truncation as SHAPE adduct detection strategies, make recommendations for SHAPE reagent choice, and outline areas for future development.

Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control.