Modulation of RNA Condensation by the DEAD-Box Protein eIF4A.

Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.

Emerging Roles for Intermolecular RNA-RNA Interactions in RNP Assemblies.

Eukaryotic cells contain large assemblies of RNA and protein, referred to as ribonucleoprotein (RNP) granules, which include cytoplasmic P-bodies, stress granules, and neuronal and germinal granules, as well as nuclear paraspeckles, Cajal bodies, and RNA foci formed from repeat expansion RNAs. Recent evidence argues that intermolecular RNA-RNA interactions play a role in forming and determining the composition of certain RNP granules. We hypothesize that intermolecular RNA-RNA interactions are favored in cells yet are limited by RNA-binding proteins, helicases, and ribosomes, thereby allowing normal RNA function. An over-abundance of intermolecular RNA-RNA interactions may be toxic since perturbations that increase RNA-RNA interactions such as long repeat expansion RNAs, arginine-containing dipeptide repeat polypeptides, and sequestration or loss of abundant RNA-binding proteins can contribute to degenerative diseases.

RNA partitioning into stress granules is based on the summation of multiple interactions.

Stress granules (SGs) are stress-induced RNA-protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple elements. Moreover, artificial tethering of G3BP1, TIA1, or FMRP can target mRNAs into SGs in a dose-dependent manner with numerous interactions required for efficient SG partitioning, which suggests individual protein interactions have small effects on the SG partitioning of mRNPs. This is supported by the observation that the SG transcriptome is largely unchanged in cell lines lacking the abundant SG RNA-binding proteins G3BP1 and G3BP2. We suggest the targeting of RNPs into SGs is due to a summation of potential RNA-protein, protein-protein, and RNA-RNA interactions with no single interaction dominating RNP recruitment into SGs.

RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome.

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein-protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein-protein and RNA-RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA-RNA assembly in cells could lead to disease.

Experimental considerations for precise RNA-mediated insertion of transgenes.

Precise RNA-mediated insertion of transgenes (PRINT) is a pioneering method for site-specific, safe-harbor transgene supplementation of the human genome that harnesses a eukaryotic retroelement protein and relies solely on the delivery of RNA. Here we outline important considerations in the design of the two required RNAs, details for the production and transfection of these RNAs to cells, and read-outs for successful transgene addition. Throughout, tips and key concepts are laid out to enable general use of this method.

Conserved and divergent DNA recognition specificities and functions of R2 retrotransposon N-terminal domains.

R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.

Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

Principles of Stress Granules Revealed by Imaging Approaches.

Eukaryotic cells contain a large number of RNA-protein assemblies, generically referred to as ribonucleoprotein (RNP) granules. Such RNP granules include stress granules and P-bodies in the cytosol and the nucleolus, Cajal bodies, and paraspeckles in the nucleus. A variety of imaging approaches have been used to reveal different components, structural features, and dynamics of RNP granules. In this review, we discuss imaging approaches that have been used to study stress granules and the insights gained from these experiments. A general theme is that these approaches can be transferred to other RNP granules to examine similar aspects of their composition, ultrastructure, dynamics and control.

Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly.

Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules. We suggest assemblies based on combinations of specific interactions and promiscuous IDRs are common features of eukaryotic cells.

Development of an Unnatural Amino Acid Incorporation System in the Actinobacterial Natural Product Producer Streptomyces venezuelae ATCC 15439.

Many Actinobacteria, most notably Streptomyces, produce structurally diverse bioactive natural products, including ribosomally synthesized peptides, by multistep enzymatic pathways. The use of site-specific genetic incorporation of unnatural amino acids to investigate and manipulate the functions of natural product biosynthetic enzymes, enzyme complexes, and ribosomally derived peptides in these organisms would have important implications for drug discovery and development efforts. Here, we have designed, constructed, and optimized unnatural amino acid systems capable of incorporating p-iodo-l-phenylalanine and p-azido-l-phenylalanine site-specifically into proteins in the model natural product producer Streptomyces venezuelae ATCC 15439. We observed notable differences in the fidelity and efficiency of these systems between S. venezuelae and previously used hosts. Our findings serve as a foundation for using an expanded genetic code in Streptomyces to address questions related to natural product biosynthesis and mechanism of action that are relevant to drug discovery and development.