The Story of RNA Unfolded: The Molecular Function of DEAD- and DExH-Box ATPases and Their Complex Relationship with Membraneless Organelles.

DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.

The Role of DEAD-Box ATPases in Gene Expression and the Regulation of RNA-Protein Condensates.

DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.

RNA granules: functional compartments or incidental condensates?

RNA granules are mesoscale assemblies that form in the absence of limiting membranes. RNA granules contain factors for RNA biogenesis and turnover and are often assumed to represent specialized compartments for RNA biochemistry. Recent evidence suggests that RNA granules assemble by phase separation of subsoluble ribonucleoprotein (RNP) complexes that partially demix from the cytoplasm or nucleoplasm. We explore the possibility that some RNA granules are nonessential condensation by-products that arise when RNP complexes exceed their solubility limit as a consequence of cellular activity, stress, or aging. We describe the use of evolutionary and mutational analyses and single-molecule techniques to distinguish functional RNA granules from "incidental condensates."

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3.

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate.

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP).

Throughout their life cycle, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Each mRNA is part of multiple successive mRNP complexes that participate in their biogenesis, cellular localization, translation and decay. The dynamic composition of mRNP complexes and their structural remodelling play crucial roles in the control of gene expression. Studying the endogenous composition of different mRNP complexes is a major challenge. In this chapter, we describe the variety of protein-centric immunoprecipitation methods available for the identification of mRNP complexes and the requirements for their experimental settings.

Methods to study circRNA-protein interactions.

Circular RNAs (circRNAs) have been studied extensively in the last few years, uncovering functional roles in a diverse range of cell types and organisms. As shown for a few cases, these functions may be mediated by trans-acting factors, in particular RNA-binding proteins (RBPs). However, the specific interaction partners for most circRNAs remain unknown. This is mainly due to technical difficulties in their identification and in differentiating between interactors of circRNAs and their linear counterparts. Here we review the currently used methodology to systematically study circRNA-protein complexes (circRNPs), focusing either on a specific RNA or protein, both on the gene-specific or global level, and discuss advantages and challenges of the available approaches.

RNA and DNA G-quadruplexes bind to human dicer and inhibit its activity.

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

Dead or alive: DEAD-box ATPases as regulators of ribonucleoprotein complex condensation.

DEAD-box ATPase proteins are found in all clades of life and have been associated with a diverse array of RNA-processing reactions in eukaryotes, bacteria and archaea. Their highly conserved core enables them to bind RNA, often in an ATP-dependent manner. In the course of the ATP hydrolysis cycle, they undergo conformational rearrangements, which enable them to unwind short RNA duplexes or remodel RNA-protein complexes. Thus, they can function as RNA helicases or chaperones. However, when their conformation is locked, they can also clamp RNA and create ATP-dependent platforms for the formation of higher-order ribonucleoprotein complexes. Recently, it was shown that DEAD-box ATPases globally regulate the phase-separation behavior of RNA-protein complexes in vitro and control the dynamics of RNA-containing membraneless organelles in both pro- and eukaryotic cells. A role of these enzymes as regulators of RNA-protein condensates, or 'condensases', suggests a unifying view of how the biochemical activities of DEAD-box ATPases are used to keep cellular condensates dynamic and 'alive', and how they regulate the composition and fate of ribonucleoprotein complexes in different RNA processing steps.

Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

Interactome analysis of the human Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (hMTr1) protein.

In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap-specific messenger RNA (mRNA) (nucleoside-2'-O-)-methyltransferase 1 (hMTr1), which catalyzes methylation of the 2'-O -ribose of the first nucleotide of capped mRNAs. Pre-mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5'-end of the whole cellular pre-mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre-mRNA maturation was identified, including splicing factors, spliceosome-associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA-binding proteins and proteins involved in mRNA 5'- and 3'-end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre-mRNA synthesis.

Diverging RNPs: Toward Understanding lncRNA-Protein Interactions and Functions.

RNA-protein interactions are essential to a variety of biological processes. The realization that mammalian genomes are pervasively transcribed brought a tidal wave of tens of thousands of newly identified long noncoding RNAs (lncRNAs) and raised questions about their purpose in cells. The vast majority of lncRNAs have yet to be studied, and it remains to be determined to how many of these transcripts a function can be ascribed. However, results gleaned from studying a handful of these macromolecules have started to reveal common themes of biological function and mechanism of action involving intricate RNA-protein interactions. Some lncRNAs were shown to regulate the chromatin and transcription of distant and neighboring genes in the nucleus, while others regulate the translation or localization of proteins in the cytoplasm. Some lncRNAs were found to be crucial during development, while mutations and aberrant expression of others have been associated with several types of cancer and a plethora of diseases. Over the last few years, the establishment of new technologies has been key in providing the tools to decode the rules governing lncRNA-protein interactions and functions. This chapter will highlight the general characteristics of lncRNAs, their function, and their mode of action, with a special focus on protein interactions. It will also describe the methods at the disposition of scientists to help them cross this next frontier in our understanding of lncRNA biology.

Site-Directed Chemical Probing to map transient RNA/protein interactions.

RNA-protein interactions are at the bases of many biological processes, forming either tight and stable functional ribonucleoprotein (RNP) complexes (i.e. the ribosome) or transitory ones, such as the complexes involving RNA chaperone proteins. To localize the sites where a protein interacts on an RNA molecule, a common simple and inexpensive biochemical method is the footprinting technique. The protein leaves its footprint on the RNA acting as a shield to protect the regions of interaction from chemical modification or cleavages obtained with chemical or enzymatic nucleases. This method has proven its efficiency to study in vitro the organization of stable RNA-protein complexes. Nevertheless, when the protein binds the RNA very dynamically, with high off-rates, protections are very often difficult to observe. For the analysis of these transient complexes, we describe an alternative strategy adapted from the Site Directed Chemical Probing (SDCP) approach and we compare it with classical footprinting. SDCP relies on the modification of the RNA binding protein to tether an RNA probe (usually Fe-EDTA) to specific protein positions. Local cleavages on the regions of interaction can be used to localize the protein and position its domains on the RNA molecule. This method has been used in the past to monitor stable complexes; we provide here a detailed protocol and a practical example of its application to the study of Escherichia coli RNA chaperone protein S1 and its transitory complexes with mRNAs.

Long noncoding RNAs in diseases of aging.

Aging is a process during which progressive deteriorating of cells, tissues, and organs over time lead to loss of function, disease, and death. Towards the goal of extending human health span, there is escalating interest in understanding the mechanisms that govern aging-associated pathologies. Adequate regulation of expression of coding and noncoding genes is critical for maintaining organism homeostasis and preventing disease processes. Long noncoding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression at all levels--transcriptional, post-transcriptional and post-translational. In this review, we discuss our emerging understanding of lncRNAs implicated in aging illnesses. We focus on diseases arising from age-driven impairment in energy metabolism (obesity, diabetes), the declining capacity to respond homeostatically to proliferative and damaging stimuli (cancer, immune dysfunction), and neurodegeneration. We identify the lncRNAs involved in these ailments and discuss the rising interest in lncRNAs as diagnostic and therapeutic targets to ameliorate age-associated pathologies and prolong health. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

SMN control of RNP assembly: from post-transcriptional gene regulation to motor neuron disease.

At the post-transcriptional level, expression of protein-coding genes is controlled by a series of RNA regulatory events including nuclear processing of primary transcripts, transport of mature mRNAs to specific cellular compartments, translation and ultimately, turnover. These processes are orchestrated through the dynamic association of mRNAs with RNA binding proteins and ribonucleoprotein (RNP) complexes. Accurate formation of RNPs in vivo is fundamentally important to cellular development and function, and its impairment often leads to human disease. The survival motor neuron (SMN) protein is key to this biological paradigm: SMN is essential for the biogenesis of various RNPs that function in mRNA processing, and genetic mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP assembly. We discuss advances in our understanding of SMN activity as a chaperone of RNPs and how disruption of SMN-dependent RNA pathways can cause motor neuron disease.

LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression.

Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60-70 days). However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6), is specifically involved in type I collagen regulation. In the 5'untranslated region (5'UTR) of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL) structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5'SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP), 25 kD FK506 binding protein (FKBP25) and RNA helicase A (RHA), contribute to this process.

Evolutionary Events Promoted Polymerase Activity of H13N8 Avian Influenza Virus.

Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.

CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding.

Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene(s). In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein (RNP) complex assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding.

Local mRNA translation and cytoskeletal reorganization: Mechanisms that tune neuronal responses.

Neurons are highly polarized cells with significantly long axonal and dendritic extensions that can reach distances up to hundreds of centimeters away from the cell bodies in higher vertebrates. Their successful formation, maintenance, and proper function highly depend on the coordination of intricate molecular networks that allow axons and dendrites to quickly process information, and respond to a continuous and diverse cascade of environmental stimuli, often without enough time for communication with the soma. Two seemingly unrelated processes, essential for these rapid responses, and thus neuronal homeostasis and plasticity, are local mRNA translation and cytoskeletal reorganization. The axonal cytoskeleton is characterized by high stability and great plasticity; two contradictory attributes that emerge from the powerful cytoskeletal rearrangement dynamics. Cytoskeletal reorganization is crucial during nervous system development and in adulthood, ensuring the establishment of proper neuronal shape and polarity, as well as regulating intracellular transport and synaptic functions. Local mRNA translation is another mechanism with a well-established role in the developing and adult nervous system. It is pivotal for axonal guidance and arborization, synaptic formation, and function and seems to be a key player in processes activated after neuronal damage. Perturbations in the regulatory pathways of local translation and cytoskeletal reorganization contribute to various pathologies with diverse clinical manifestations, ranging from intellectual disabilities (ID) to autism spectrum disorders (ASD) and schizophrenia (SCZ). Despite the fact that both processes are essential for the orchestration of pathways critical for proper axonal and dendritic function, the interplay between them remains elusive. Here we review our current knowledge on the molecular mechanisms and specific interaction networks that regulate and potentially coordinate these interconnected processes.

Analyzing the Composition and Organization of Ribonucleoprotein Complexes by APEX-Seq.

Diverse protein-RNA complexes assemble in cells, and their composition and localization regulate the fate of mRNAs. Here, we detail APEX-Seq, an experimental strategy to capture protein-RNA interactions and profile their sub-cellular organization by in vivo proximity labeling and high-throughput sequencing. APEX-Seq relies on direct proximity labeling of RNAs by the peroxidase enzyme APEX2, which can be targeted to specific sites in the cell or fused to proteins of interest. Direct RNA proximity labeling promises new insights into the dynamic behavior of RNA, addressing length scales beyond direct physical contact but too short for microscopy. APEX-Seq should be widely applicable to diverse biological questions and in many cell types, enabling comprehensive studies of the spatial transcriptome and its dynamics over time.