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."

Germ granules in development.

A hallmark of all germ cells is the presence of germ granules: assemblies of proteins and RNA that lack a delineating membrane and are proposed to form via condensation. Germ granules across organisms share several conserved components, including factors required for germ cell fate determination and maintenance, and are thought to be linked to germ cell development. The molecular functions of germ granules, however, remain incompletely understood. In this Development at a Glance article, we survey germ granules across organisms and developmental stages, and highlight emerging themes regarding granule regulation, dynamics and proposed functions.

Implications of methodologies for integrating empirical kinships into ex situ population management using PMx: A case study of Baer's Pochard (Aythya baeri) in North America.

The application of molecular tools to population management can improve the long-term genetic viability of ex situ populations. In this study, we aimed to understand the implications of integrating empirical kinships into the genetic management of an ex situ population of the endangered waterfowl, Baer's pochard (Aythya baeri), in North America. Single nucleotide polymorphism data were generated for 141 Baer's pochard using double digest restriction site-associated DNA sequencing and empirical kinships were derived and integrated into the population management software PMx. Analyses suggested 37.7% of pairwise relationships previously assumed to be unrelated were first, second, or third-order relatives. We determined that most genetic summary statistics were impacted through the calculation of the population's mean kinship, which increased from MK¯=0.0772 to MK¯=0.2074 after empirical kinships were integrated into our analyses. Our results also revealed the importance of understanding how molecular kinships derived from a particular estimator are scaled, if the scale differs significantly from pedigree-based kinships. We describe the theory behind the genetic metrics impacted and provide general guidance on incorporating empirical kinships into ex situ population management as well as provide suggestions for sampling strategies to minimize the biases inherent in merging two types of kinship estimators.

Division of Labor in an Oligomer of the DEAD-Box RNA Helicase Ded1p.

Most aspects of RNA metabolism involve DEAD-box RNA helicases, enzymes that bind and remodel RNA and RNA-protein complexes in an ATP-dependent manner. Here we show that the DEAD-box helicase Ded1p oligomerizes in the cell and in vitro, and unwinds RNA as a trimer. Two protomers bind the single-stranded region of RNA substrates and load a third protomer to the duplex, which then separates the strands. ATP utilization differs between the strand-separating protomer and those bound to the single-stranded region. Binding of the eukaryotic initiation factor 4G to Ded1p interferes with oligomerization and thereby modulates unwinding activity and RNA affinity of the helicase. Our data reveal a strict division of labor between the Ded1p protomers in the oligomer. This mode of oligomerization fundamentally differs from other helicases. Oligomerization represents a previously unappreciated level of regulation for DEAD-box helicase activities.

Ex situ breeding programs benefit from science-based cooperative management.

Science-based management confers a variety of benefits to wildlife populations that are cooperatively managed by zoos and aquariums, including those managed through the Association of Zoos and Aquariums. Briefly, when management strategies are successful, they result in reproductively robust populations that better retain genetic diversity and limit inbreeding than unmanaged populations. Although the benefits of demographic and genetic management have been well documented throughout both the scientific and popular literature, it has also been established that the majority of managed populations in zoos and aquariums are not meeting the minimum criteria believed to convey long-term biological viability. For most of these populations, an inability to meet viability criteria is not an inherent failure of how cooperative management is implemented. Furthermore, in recent years, we have perceived that the need to meet specific viability goals sometimes has obscured the benefits that these populations receive from rigorous, science-based management. To better clarify the conversation surrounding population viability in zoos and aquariums, we seek to decouple viability measures and how they predict population persistence from the benefits conferred to populations through science-based management. A primary goal of population management is to facilitate the persistence of priority species for longer than would be expected if no such management were implemented. Although current viability measures and future projections of viability are important tools for assessing the likelihood of population persistence, they are not indicators of which populations may most benefit from science-based management. Here, we review the history and purpose of applying science-based management to zoo and aquarium populations, describe measures of population viability and caution against confusing those measures of viability with population management goals or long-term population sustainability, and clearly articulate the benefits conferred to zoo and aquarium populations by science-based management.

A comparative study of small molecules targeting eIF4A.

The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity-generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.

Assessing possible hybridization among managed Nubian ibex in North America.

Hybridization among closely related species is a concern in zoo and aquarium populations where unpedigreed animals are frequently exchanged with the private sector. In this study, we examine possible hybridization in a group of Nubian ibex (Capra nubiana) imported into the Association of Zoos and Aquariums' (AZA) Species Survival Program (SSP) from a private institution. These individuals appeared smaller in stature than adult SSP Nubian ibex and were excluded from breeding recommendations over the concern that they were hybrids. Twenty-six microsatellites were used to rule out recent hybridization with domestic goats, Siberian ibex (Capra sibirica), and Alpine ibex (Capra ibex). We argue that natural phenotypic variation across the large geographic range of Nubian ibex may account for the small stature of the imported ibex, as private institutions may have historically acquired individuals from locations that differed from the SSP founders. However, the imported Nubian ibex appeared genetically differentiated from the SSP Nubian ibex and may represent a source of genetic variation for the managed population.

Use of molecular data in zoo and aquarium collection management: Benefits, challenges, and best practices.

The global zoo and aquarium community widely recognizes that its animal collections and cooperative breeding programs are facing a sustainability crisis. It has become commonly accepted that numerous priority species cannot be maintained unless new management strategies are adopted. While molecular data have the potential to greatly improve management across a range of scenarios, they have been generally underutilized by the zoo and aquarium community. This failure to effectively apply molecular data to collection management has been due, in part, to a paucity of resources within the community on which to base informed decisions about when the use of such data is appropriate and what steps are necessary to successfully integrate data into management. Here, we identify three broad areas of inquiry where molecular data can inform management: 1) taxonomic identification; 2) incomplete or unknown pedigrees; and 3) hereditary disease. Across these topics, we offer a discussion of the advantages, limitations, and considerations for applying molecular data to ex situ animal populations in a style accessible to zoo and aquarium professionals. Ultimately, we intend for this compiled information to serve as a resource for the community to help ensure that molecular projects directly and effectively benefit the long-term persistence of ex situ populations.

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE.

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75's complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination.

Applying SNP-Derived Molecular Coancestry Estimates to Captive Breeding Programs.

Captive breeding programs for wildlife species typically rely on pedigrees to inform genetic management. Although pedigree-based breeding strategies are quite effective at retaining long-term genetic variation, management of zoo-based breeding programs continues to be hampered when pedigrees are poorly known. The objective of this study was to evaluate 2 options for generating single nucleotide polymorphism (SNP) data to resolve unknown relationships within captive breeding programs. We generated SNP data for a zoo-based population of addax (Addax nasomasculatus) using both the Illumina BovineHD BeadChip and double digest restriction site-associated DNA (ddRAD) sequencing. Our results demonstrated that estimates of allele sharing (AS) between pairs of individuals exhibited low variances. Average AS variances were highest when using 50 loci (SNPchipall = 0.00159; ddRADall = 0.0249), but fell below 0.0003 for the SNP chip dataset when sampling ≥250 loci and below 0.0025 for the ddRAD dataset when sampling ≥500 loci. Furthermore, the correlation between the SNPchipall and ddRADall AS datasets was 0.88 (95%CI = 0.84-0.91) when subsampling 500 loci. Collectively, our results indicated that both SNP genotyping methods produced sufficient data for accurately estimating relationships, even within an extremely bottlenecked population. Our results also suggested that analytic assumptions historically integrated into the addax pedigree are not adversely impacting long-term pedigree-based management; kinships calculated from the analytic pedigree were significantly correlated (P << 0.001) with AS estimates. Overall, our conclusions are intended to serve as both a proof of concept and a model for applying molecular data to the genetic management of captive breeding programs.

Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability.

Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)(+) RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype.

DEAD-box helicases form nucleotide-dependent, long-lived complexes with RNA.

DEAD-box RNA helicases bind and remodel RNA and RNA-protein complexes in an ATP-dependent fashion. Several lines of evidence suggest that DEAD-box RNA helicases can also form stable, persistent complexes with RNA in a process referred to as RNA clamping. The molecular basis of RNA clamping is not well understood. Here we show that the yeast DEAD-box helicase Ded1p forms exceptionally long-lived complexes with RNA and the nonhydrolyzable ATP ground-state analogue ADP-BeFx or the nonhydrolyzable ATP transition state analogue ADP-AlFx. The complexes have lifetimes of several hours, and neither nucleotide nor Mg(2+) is released during this period. Mutation of arginine 489, which stabilizes the transition state, prevents formation of long-lived complexes with the ATP transition state analogue, but not with the ground state analogue. We also show that two other yeast DEAD-box helicases, Mss116p and Sub2p, form comparably long-lived complexes with RNA and ADP-BeFx. Like Ded1p, Mss116p forms long-lived complexes with ADP-AlFx, but Sub2p does not. These data suggest that the ATP transition state might vary for distinct DEAD-box helicases, or that the transition state triggers differing RNA binding properties in these proteins. In the ATP ground state, however, all tested DEAD-box helicases establish a persistent grip on RNA, revealing an inherent capacity of the enzymes to function as potent, ATP-dependent RNA clamps.

DEAD-box helicases as integrators of RNA, nucleotide and protein binding.

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Kinship-based management strategies for captive breeding programs when pedigrees are unknown or uncertain.

Zoo-based captive breeding programs typically rely on accurate pedigrees to maintain long-term population genetic diversity and prevent close inbreeding. For many mixed-sex captive populations, it is difficult to assign parentage of offspring with certainty without conducting DNA-based parentage analyses. Using the demographic parameters of a North American captive population of Arabian oryx (Oryx leucoryx), 2 kinship-based breeding-pair selection strategies were modeled for their performance in handling pedigrees with varying degrees of parentage uncertainty. We also compared these strategies with 2 nonkinship-based methods. Pedigrees simulated under different management strategies were compared for their long-term ability to maintain gene diversity (GD) and avoid inbreeding. For the Arabian oryx, results indicate that recording multiple possible parents instead of removing the unknown genomic portion of the pedigree can more efficiently utilize all animals available for breeding without compromising GD and inbreeding avoidance. Both kinship-based breeding-pair selection strategies significantly outperformed the nonkinship-based strategies.

High-resolution fleezers reveal duplex opening and stepwise assembly by an oligomer of the DEAD-box helicase Ded1p.

DEAD-box RNA helicases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box helicases unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened. Using high-resolution optical tweezers and fluorescence, we reveal a highly dynamic and stochastic process of multiple Ded1p protomers assembling on and unwinding an RNA duplex. One Ded1p protomer binds to a duplex-adjacent ssRNA tail and promotes binding and subsequent unwinding of the duplex by additional Ded1p protomers in 4-6 bp steps. The data also reveal rapid duplex unwinding and rezipping linked with binding and dissociation of individual protomers and coordinated with the ATP hydrolysis cycle.

The P granule antibody KT3 recognizes epitopes in both PGL-1 and PGL-3.

The KT3 antibody is a commercially available antibody that recognizes the P granule protein PGL-3 (Takeda et al., 2008). Using immunostaining and western blotting of purified peptide fragments, we show that KT3 recognizes both PGL-3 and its paralog PGL-1 , likely through a shared epitope in the intrinsically disordered region.

DDX3X and Stress Granules: Emerging Players in Cancer and Drug Resistance.

The DEAD (Asp-Glu-Ala-Asp)-box helicase 3 X-linked (DDX3X) protein participates in many aspects of mRNA metabolism and stress granule (SG) formation. DDX3X has also been associated with signal transduction and cell cycle regulation that are important in maintaining cellular homeostasis. Malfunctions of DDX3X have been implicated in multiple cancers, including brain cancer, leukemia, prostate cancer, and head and neck cancer. Recently, literature has reported SG-associated cancer drug resistance, which correlates with a negative disease prognosis. Based on the connections between DDX3X, SG formation, and cancer pathology, targeting DDX3X may be a promising direction for cancer therapeutics development. In this review, we describe the biological functions of DDX3X in terms of mRNA metabolism, signal transduction, and cell cycle regulation. Furthermore, we summarize the contributions of DDX3X in SG formation and cellular stress adaptation. Finally, we discuss the relationships of DDX3X, SG, and cancer drug resistance, and discuss the current research progress of several DDX3X inhibitors for cancer treatment.

Comparing genome-based estimates of relatedness for use in pedigree-based conservation management.

Researchers have long debated which estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the methods used to generate markers, marker quality, and levels of diversity in sampled individuals. Here, we compare six commonly used genome-based relatedness estimators (kinship genetic distance [KGD], Wang maximum likelihood [TrioML], Queller and Goodnight [Rxy ], Kinship INference for Genome-wide association studies [KING-robust), and pairwise relatedness [RAB ], allele-sharing coancestry [AS]) across five species bred in captivity-including three birds and two mammals-with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data. Genome-based relatedness estimates varied widely across estimators, sequencing methods, and species, yet the most consistent results for known first order relationships were found using Rxy , RAB , and AS. However, AS was found to be less consistently correlated with known pedigree relatedness than either Rxy or RAB . Our combined results indicate there is not a single genome-based estimator that is ideal across different species and data types. To determine the most appropriate genome-based relatedness estimator for each new data set, we recommend assessing the relative: (1) correlation of candidate estimators with known relationships in the pedigree and (2) precision of candidate estimators with known first-order relationships. These recommendations are broadly applicable to conservation breeding programmes, particularly where genome-based estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in the application of wild pedigrees, our results are also applicable to in situ wildlife management.

ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding.

DEAD-box proteins, the largest helicase family, catalyze ATP-dependent remodeling of RNA-protein complexes and the unwinding of RNA duplexes. Because DEAD-box proteins hydrolyze ATP in an RNA-dependent fashion, the energy provided by ATP hydrolysis is commonly assumed to drive the energetically unfavorable duplex unwinding. Here, we show efficient unwinding of stable duplexes by several DEAD-box proteins in the presence of the nonhydrolyzable ATP analog ADP-beryllium fluoride. Another ATP analog, ADP-aluminum fluoride, does not promote unwinding. The findings show that the energy from ATP hydrolysis is dispensable for strand separation. ATP binding, however, appears necessary. ATP hydrolysis is found to be required for fast enzyme release from the RNA and multiple substrate turnovers and thus for enzyme recycling.

Cell-free reconstitution of multi-condensate assemblies.

Biomolecular condensates (BCs) are intracellular condensates that form by phase separation of proteins and RNA from the nucleoplasm or cytoplasm. BCs often form complex assemblies where compositionally distinct condensates wet each other without mixing. In this chapter, we describe methods to reconstitute multi-condensate assemblies from purified components. We include protocols to express, purify, label, and analyze the dynamics of proteins and RNAs that drive multi-condensate assembly. Analysis of the condensation and wetting behaviors of condensates in cell-free reconstituted systems can be used to define the molecular interactions that regulate BCs in cells.