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Arginine–glycine–glycine motif proteins play a crucial role in determining mRNA fate. Suppressor of clathrin deficiency 6 (Scd6) is a conserved arginine–glycine–glycine motif containing ribonucleoprotein (RNP) condensate–resident, translation repressor, and decapping activator protein in Saccharomyces cerevisiae. Identifying protein factors that can modulate Scd6 function is critical to understanding the regulation of mRNA fate by Scd6. In this study, using an approach that combined mRNA tethering assay with flow cytometry, we screened 50 genes for their role in modulating the translation repression activity of Scd6. We identified 8 conserved modulators with human homologs. Of these, we further characterized in detail guanine nucleotide exchange factor Rho1 multicopy suppressor 2 (Rom2) and glycolytic enzyme triose phosphate dehydrogenase 3 (Tdh3), which, respectively, impede and promote translation repression activity of Scd6. Our study reveals that Rom2 negatively regulates the arginine methylation of Scd6 and antagonizes its localization to P-bodies. Tdh3 , on the other hand, promotes Scd6 interaction with Hmt1 , thereby promoting the arginine methylation of Scd6 and enhanced eIF4G1 interaction, which is known to promote its repression activity. Identifying these novel modulators provides exciting new insights into the role of a metabolic enzyme of the glycolytic pathway and guanine nucleotide exchange factor implicated in the cell wall integrity pathway in regulating Scd6 function and, thereby, cytoplasmic mRNA fate. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are dynamic membraneless organelles that compartmentalize proteins and RNA molecules to regulate key cellular processes. Diverse RNA species exert their effects on the cell by their roles in condensate formation and function. RNA abnormalities such as overexpression, modification, and mislocalization can lead to pathological condensate behaviors that drive various diseases, including cancer, neurological disorders, and infections. Here, we review RNA's role in condensate biology, describe the mechanisms of RNA-induced condensate dysregulation, note the implications for disease pathogenesis, and discuss novel therapeutic strategies. Emerging approaches to targeting RNA within condensates, including small molecules and RNA-based therapies that leverage the unique properties of condensates, may revolutionize treatment for complex diseases. [Display omitted] Han et al. review the role of RNA in biomolecular condensates, highlighting how RNA-induced dysregulation contributes to disease pathogenesis. They discuss emerging therapeutic strategies targeting RNA within condensates, which could revolutionize treatment for complex diseases like cancer and neurological disorders. [ABSTRACT FROM AUTHOR]

Cellular stress response pathways (CSRPs) constantly monitor a changing microenvironment. Intrinsic and extrinsic stresses activate specific CSRPs that converge at the level of RNA metabolism, aiding cell survival. RNA G-quadruplexes (rG4s) are emerging stress-responsive cis -elements found in both the coding and noncoding transcriptomes. The dynamics of rG4s contribute to different aspects of RNA metabolism from transcription to decay. Cellular stress affects rG4s' dynamics and the functions of rG4-bearing transcripts. The rG4 proteome is highly enriched with RNA-binding proteins (RBPs) that contribute to cellular stress responses. Multiple RBPs bind rG4 transcripts and modulate their localization, stability, and translation in a stress-responsive manner. Interactions between rG4 transcripts and RBPs contribute to liquid–liquid phase separation (LLPS), the phenomenon that underlies the formation of membraneless compartments. Specifically, rG4s are implicated in the formation and dynamics of stress-responsive RNA granules, such as stress granules. Stress-induced rG4s are potential therapeutic targets for human disease. RNA G-quadruplexes (rG4s) are noncanonical secondary structures formed by guanine-rich sequences that are found in different regions of RNA molecules. These structures have been implicated in diverse biological processes, including translation, splicing, and RNA stability. Recent studies have suggested that rG4s play a role in the cellular response to stress. This review summarizes the current knowledge on rG4s under stress, focusing on their formation, regulation, and potential functions in stress response pathways. We discuss the molecular mechanisms that regulate the formation of rG4 under different stress conditions and the impact of these structures on RNA metabolism, gene expression, and cell survival. Finally, we highlight the potential therapeutic implications of targeting rG4s for the treatment of stress-related diseases through modulating cell survival. [ABSTRACT FROM AUTHOR]

A defining feature of a productive viral infection is the co-opting of host cell resources for viral replication. Despite the host repertoire of molecular functions and biological counter measures, viruses still subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life, forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. As such, the hallmark of the viral takeover of a cell is the reshaping of RNA fate to modulate host gene expression and evade immune responses by altering RBP interactions. Here, we provide an extensive review of work in this area, particularly on the duality of the formation of RNP complexes that can be either pro- or antiviral. Overall, in this review, we highlight the various ways viruses co-opt RBPs to regulate RNA stability and modulate the outcome of infection by gathering novel insights gained from research studies in this field. [ABSTRACT FROM AUTHOR]

A defining feature of a productive viral infection is the co-opting of host cell resources for viral replication. Despite the host repertoire of molecular functions and biological counter measures, viruses still subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life, forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. As such, the hallmark of the viral takeover of a cell is the reshaping of RNA fate to modulate host gene expression and evade immune responses by altering RBP interactions. Here, we provide an extensive review of work in this area, particularly on the duality of the formation of RNP complexes that can be either pro- or antiviral. Overall, in this review, we highlight the various ways viruses co-opt RBPs to regulate RNA stability and modulate the outcome of infection by gathering novel insights gained from research studies in this field.

Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca2+-regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure.Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3′UTRs as well as c-myc 5′UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the KD of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3′UTR with KDs in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5′UTR indicating some selectivity.Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca2+. The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context.

RNA is a vital biomolecule, the function of which is tightly spatiotemporally regulated. RNA organelles are biological structures that either membrane-less or surrounded by membrane. They are produced by the all the cells and indulge in vital cellular mechanisms. They include the intracellular RNA granules and the extracellular exosomes. RNA granules play an essential role in intracellular regulation of RNA localization, stability and translation. Aberrant regulation of RNA is connected to disease development. For example, in microsatellite diseases such as CXG repeat expansion disorders, the mutant CXG repeat RNA’s localization and function are affected. RNA is not only transported intracellularly but can also be transported between cells via exosomes. The loading of the exosomes is regulated by RNA-protein complexes, and recent studies show that cytosolic RNA granules and exosomes share common content. Intracellular RNA granules and exosome loading may therefore be related. Exosomes can also transfer pathogenic molecules of CXG diseases from cell to cell, thereby driving disease progression. Both intracellular RNA granules and extracellular RNA vesicles may serve as a source for diagnostic and treatment strategies. In therapeutic approaches, pharmaceutical agents may be loaded into exosomes which then transport them to the desired cells/tissues. This is a promising target specific treatment strategy with few side effects. With respect to diagnostics, disease-specific content of exosomes, e.g., RNA-signatures, can serve as attractive biomarker of central nervous system diseases detecting early physiological disturbances, even before symptoms of neurodegeneration appear and irreparable damage to the nervous system occurs. In this review, we summarize the known function of cytoplasmic RNA granules and extracellular vesicles, as well as their role and dysfunction in CXG repeat expansion disorders. We also provide a summary of established protocols for the isolation and characterization of both cytoplasmic and extracellular RNA organelles.

RNA imaging is of great importance for understanding its complex spatiotemporal dynamics and cellular functions. Considerable effort has been devoted to the development of small-molecule fluorescent probes for RNA imaging. However, most of the reported studies have mainly focused on improving the photostability, permeability, long emission wavelength, and compatibility with live-cell imaging of RNA probes. Less attention has been paid to the selectivity and detection limit of this class of probes. Highly selective and sensitive RNA probes are still rarely available. In this study, a new set of styryl probes were designed and synthesized, with the aim of upgrading the detection limit and maintaining the selectivity of a lead probe QUID−1 for RNA. Among these newly synthesized compounds, QUID−2 was the most promising candidate. The limit of detection (LOD) value of QUID−2 for the RNA was up to 1.8 ng/mL in solution. This property was significantly improved in comparison with that of QUID−1. Further spectroscopy and cell imaging studies demonstrated the advantages of QUID−2 over a commercially available RNA staining probe, SYTO RNASelect, for highly selective and sensitive RNA imaging. In addition, QUID−2 exhibited excellent photostability and low cytotoxicity. Using QUID−2, the global dynamics of RNA were revealed in live cells. More importantly, QUID−2 was found to be potentially applicable for detecting RNA granules in live cells. Collectively, our work provides an ideal probe for RNA imaging. We anticipate that this powerful tool may create new opportunities to investigate the underlying roles of RNA and RNA granules in live cells. [ABSTRACT FROM AUTHOR]

Cells regulate protein synthesis under stressful circumstances by forming cytoplasmic RNA granules, termed stress granules (SGs). SGs are membrane-less organelles that function as a protective mechanism in response to stress. They function through liquid-liquid phase separation, which is a vital process comprising 2 distinct de-mixed liquid phases. The components of SGs, such as G3BP1, can serve as biomarkers of cell toxicity. Respiratory diseases are among the leading causes of death globally. After the humidifier disinfectant disaster in Korea in 2011, social concerns over respiratory disease-related deaths have been raised, and the importance of inhalation toxicity testing has been emphasized. Traditionally, in vivo animal models have been used to assess inhalation toxicity, but these models still have limitations owing to physiological differences between species. To overcome these limitations, human immortalized lung epithelial and lung cancer cell lines have been used to evaluate lung toxicity in vitro. Human stem cell-derived 3-dimensional organoid technology has recently been developed in various research fields, including lung toxicity. This review discusses SG-related proteins as potential biomarkers for lung toxicity assessment, especially in human lung organoids under stress conditions, such as exposure to toxic chemicals.

Disordered RNA-binding proteins and repetitive RNA sequences are the main genetic causes of several neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington’s disease. Importantly, these components also seed the formation of cytoplasmic liquid-like granules, like stress granules and P bodies. Emerging evidence demonstrates that healthy granules formed via liquid-liquid phase separation can mature into solid- or gel-like inclusions that persist within the cell. These solidified inclusions are a precursor to the aggregates identified in patients, demonstrating that dysregulation of RNA granule biology is an important component of neurodegeneration. Here, we review recent literature highlighting how RNA molecules seed proteinaceous granules, the mechanisms of healthy turnover of RNA granules in cells, which biophysical properties underly a transition to solid- or gel-like material states, and why persistent granules disrupt the cellular homeostasis of neurons. We also identify various methods that will illuminate the contributions of disordered proteins and RNAs to neurodegeneration in ongoing research efforts.

Most of the human genome, except for a small region that transcribes protein coding RNAs, was considered junk. With the advent of RNA sequencing technology, we know that much of the genome codes for RNAs with no protein-coding potential. Long non-coding RNAs (lncRNAs) that form a significant proportion are dynamically expressed and play diverse roles in physiological and pathological processes. Precise spatiotemporal control of their expression is essential to carry out various biochemical reactions inside the cell. Intracellular organelles with membrane-bound compartments are known for creating an independent internal environment for carrying out specific functions. The formation of membrane-free ribonucleoprotein condensates resulting in intracellular compartments is documented in recent times to execute specialized tasks such as DNA replication and repair, chromatin re modeling, transcription, and mRNA splicing. These liquid compartments, called membrane-less organelles (MLOs), are formed by liquid–liquid phase separation (LLPS), selectively partitioning a specific set of macromolecules from others. While RNA binding proteins (RBPs) with low complexity regions (LCRs) appear to play an essential role in this process, the role of RNAs is not well understood. It appears that short nonspecific RNAs keep the RBPs in a soluble state, while longer RNAs with unique secondary structures promote LLPS formation by specifically binding to RBPs. This review will update the current understanding of phase separation, physio-chemical nature and composition of condensates, regulation of phase separation, the role of lncRNA in the phase separation process, and the relevance to cancer development and progression. [ABSTRACT FROM AUTHOR]

Stress-induced condensation of mRNA and protein into massive cytosolic clusters is conserved across eukaryotes. Known as stress granules when visible by imaging, these structures remarkably have no broadly accepted biological function, mechanism of formation or dispersal, or even molecular composition. As part of a larger surge of interest in biomolecular condensation, studies of stress granules and related RNA/protein condensates have increasingly probed the biochemical underpinnings of condensation. Here, we review open questions and recent advances, including the stages from initial condensate formation to accumulation in mature stress granules, mechanisms by which stress-induced condensates form and dissolve, and surprising twists in understanding the RNA components of stress granules and their role in condensation. We outline grand challenges in understanding stress-induced RNA condensation, centering on the unique and substantial barriers in the molecular study of cellular structures, such as stress granules, for which no biological function has been firmly established. [Display omitted] Stress granules (SGs), canonical membraneless organelles, have seen a surge of activity in identifying their protein/RNA components and biophysical underpinnings. Glauninger et al. review our understanding of SGs and their biomolecular condensate precursors, emphasizing conceptual and methodological challenges arising from the lack of established biological functions for these conserved structures. [ABSTRACT FROM AUTHOR]

Since its initial characterization in 2016, the interferon stimulated gene Shiftless (SHFL) has proven to be a critical piece of the innate immune response to viral infection. SHFL expression stringently restricts the replication of multiple DNA, RNA, and retroviruses with an extraordinary diversity of mechanisms that differ from one virus to the next. These inhibitory strategies include the negative regulation of viral RNA stability, translation, and even the manipulation of RNA granule formation during viral infection. Even more surprisingly, SHFL is the first human protein found to directly inhibit the activity of the -1 programmed ribosomal frameshift, a translation recoding strategy utilized across nearly all domains of life and several human viruses. Recent literature has shown that SHFL expression also significantly impacts viral pathogenesis in mouse models, highlighting its in vivo efficacy. To help reconcile the many mechanisms by which SHFL restricts viral replication, we provide here a comprehensive review of this complex ISG, its influence over viral RNA fate, and the implications of its functions on the virus-host arms race for control of the cell. [ABSTRACT FROM AUTHOR]

Numerous membrane‐less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super‐enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane‐less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules. Synopsis: It is unclear whether RNA is required for the integrity of all the diverse types of RNP granules found in the nucleus and cytoplasm of eukaryotic cells. Here, degradation of intracellular RNA in either the cytosol or the nucleus is found to alter the majority of membrane‐less RNA protein granules, indicating a widespread RNA role in their organization. Loss of cytoplasmic RNA has a differential effect on stress granules and processing bodies, where P‐bodies are largely not altered.Targeting RNase L to the nucleus provides an inducible means to degrade nuclear RNA.Loss of nuclear RNA alters the assembly of the nucleolus, nuclear speckles, and Cajal bodies.Loss of nuclear RNA does not alter the size or number of transcriptional condensates. [ABSTRACT FROM AUTHOR]

In Neurospora crassa, expression from an unpaired gene is suppressed by a mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNA interference (RNAi) factors to silence target mRNAs. Here, we report that Neurospora CAR-1 and CGH-1, homologs of two Caenorhabditis elegans RNA granule components, are involved in MSUD. These fungal proteins are found in the perinuclear region and P-bodies, much like their worm counterparts. They interact with components of the meiotic silencing complex (MSC), including the SMS-2 Argonaute. This is the first time MSUD has been linked to RNA granule proteins. [ABSTRACT FROM AUTHOR]

Messenger RNA is complexed with proteins throughout its life cycle. The first mRNA-containing particles of non-ribosomal nature, named informosomes, were discovered in cytoplasmic extracts of fish embryos by the laboratory of Alexander Spirin, and later described in live cells. Over time, various other nuclear and cytoplasmic mRNA-containing ribonucleoproteins (mRNPs) have been found and characterized. Although these mRNPs are very diverse in their subcellular localization, structure and functions, they share many common characteristics with informosomes. In this mini-review, I will discuss the discovery of informosomes, their characteristics and proposed functions, and their potential relationship to other mRNPs. [ABSTRACT FROM AUTHOR]

RNA imaging is of great importance for understanding its complex spatiotemporal dynamics and cellular functions. Considerable effort has been devoted to the development of small-molecule fluorescent probes for RNA imaging. However, most of the reported studies have mainly focused on improving the photostability, permeability, long emission wavelength, and compatibility with live-cell imaging of RNA probes. Less attention has been paid to the selectivity and detection limit of this class of probes. Highly selective and sensitive RNA probes are still rarely available. In this study, a new set of styryl probes were designed and synthesized, with the aim of upgrading the detection limit and maintaining the selectivity of a lead probe QUID−1 for RNA. Among these newly synthesized compounds, QUID−2 was the most promising candidate. The limit of detection (LOD) value of QUID−2 for the RNA was up to 1.8 ng/mL in solution. This property was significantly improved in comparison with that of QUID−1. Further spectroscopy and cell imaging studies demonstrated the advantages of QUID−2 over a commercially available RNA staining probe, SYTO RNASelect, for highly selective and sensitive RNA imaging. In addition, QUID−2 exhibited excellent photostability and low cytotoxicity. Using QUID−2, the global dynamics of RNA were revealed in live cells. More importantly, QUID−2 was found to be potentially applicable for detecting RNA granules in live cells. Collectively, our work provides an ideal probe for RNA imaging. We anticipate that this powerful tool may create new opportunities to investigate the underlying roles of RNA and RNA granules in live cells.

Cellular liquid–liquid phase separation is a physiologically inevitable phenomenon in molecularly crowded environments inside cells and serves to compartmentalize biomolecules to facilitate several functions, forming cytoplasmic and nuclear RNA granules. Abnormalities in the phase separation process in RNA granules are implicated in the onset of several neurodegenerative diseases; the initial liquid-like phase-separated droplets containing pathogenic proteins are prone to aberrantly mature into solid-like droplets. RNAs are involved in the maturation of physiological and pathological RNA granules and are essential for governing the fate of phase-transition processes. Notably, RNA G-quadruplex (G4RNA), which is the secondary structure of nucleic acids that are formed in guanine-rich sequences, appears to be an advantageous scaffold for RNA-derived phase separation because of its multivalent interactions with RNAs and RNA-binding proteins. Here, we summarize the properties of RNA granules in physiological and pathological phase separation and discuss the potential roles of G4RNA in granules. [ABSTRACT FROM AUTHOR]

Given the projected increase in multidrug-resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life cycle stages not targeted by drugs currently in use. Host-targeting compounds are of particular interest because they can offer a high barrier to resistance. Here, we report identification of two related small molecules that inhibit HIV-1 late events, a part of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular hostcatalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and peripheral blood mononuclear cells, and are effective against a primary isolate. They reduce virus production, likely by inhibiting a posttranslational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed upon parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that form during immature HIV-1 capsid assembly. Indeed, imaging of infected cells shows compound colocalized with two host enzymes found in assembly intermediates, ABCE1 and DDX6, but not two host proteins found in other complexes. While the exact target and mechanism of action of this chemotype remain to be determined, our findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly. IMPORTANCE The success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including virus-host interactions that promote assembly. This effort led to the identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations, likely by acting on assembly. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly. However, resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype represents a first-in-class inhibitor of virus production that acts by targeting a virus-host complex important for HIV-1 Gag assembly. [ABSTRACT FROM AUTHOR]

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi, the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation.

RNA granules are protein/RNA condensates. How specific mRNAs are recruited to cytoplasmic RNA granules is not known. Here, we characterize the transcriptome and assembly of P granules, RNA granules in the C. elegans germ plasm. We find that P granules recruit mRNAs by condensation with the disordered protein MEG-3. MEG-3 traps mRNAs into non-dynamic condensates in vitro and binds to ~500 mRNAs in vivo in a sequence-independent manner that favors embryonic mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. Localization to P granules is not required for translational repression but is required to enrich mRNAs in the germ lineage for robust germline development. Our observations reveal similarities between P granules and stress granules and identify intrinsically-disordered proteins as drivers of RNA condensation during P granule assembly.

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi , the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation. [ABSTRACT FROM AUTHOR]

The fate of messenger RNA in cytoplasm plays a crucial role in various cellular processes. However, the mechanisms that decide whether mRNA will be translated, degraded or stored remain unclear. Single stranded nucleic acid binding protein (Sbp1), an Arginine‐Glycine‐Glycine (RGG‐motif) protein, is known to promote transition of mRNA into a repressed state by binding eukaryotic translation initiation factor 4G1 (eIF4G1) and to promote mRNA decapping, perhaps by modulation of Dcp1/2 activity. Sbp1 is known to be methylated on arginine residues in RGG‐motif; however, the functional relevance of this modification in vivo remains unknown. Here, we report that Sbp1 is arginine‐methylated in an hnRNP methyl transferase (Hmt1)‐dependent manner and that methylation is enhanced upon glucose deprivation. Characterization of an arginine‐methylation‐defective (AMD) mutant provided evidence that methylation affects Sbp1 function in vivo. The AMD mutant is compromised in causing growth defect upon overexpression, and the mutant is defective in both localizing to and inducing granule formation. Importantly, the Sbp1‐eIF4G1 interaction is compromised both for the AMD mutant and in the absence of Hmt1. Upon overexpression, wild‐type Sbp1 increases localization of another RGG motif containing protein, Scd6 (suppressor of clathrin deficiency) to granules; however, this property of Sbp1 is compromised in the AMD mutant and in the absence of Hmt1, indicating that Sbp1 repression activity could involve other RGG‐motif translation repressors. Additionally, the AMD mutant fails to increase localization of the decapping activator DEAD box helicase homolog to foci and fails to rescue the decapping defect of a dcp1‐2Δski8 strain, highlighting the role of Sbp1 methylation in decapping. Taken together, these results suggest that arginine methylation modulates Sbp1 role in mRNA fate determination. [ABSTRACT FROM AUTHOR]

The hnRNP A/B family of proteins is canonically central to cellular RNA metabolism, but due to their highly conserved nature, the functional differences between hnRNP A1, A2/B1, A0, and A3 are often overlooked. In this review, we explore and identify the shared and disparate homeostatic and disease-related functions of the hnRNP A/B family proteins, highlighting areas where the proteins have not been clearly differentiated. Herein, we provide a comprehensive assembly of the literature on these proteins. We find that there are critical gaps in our grasp of A/B proteins’ alternative splice isoforms, structures, regulation, and tissue and cell-type-specific functions, and propose that future mechanistic research integrating multiple A/B proteins will significantly improve our understanding of how this essential protein family contributes to cell homeostasis and disease.

Regulation of RNA homeostasis or “RNAstasis” is a central step in eukaryotic gene expression. From transcription to decay, cellular messenger RNAs (mRNAs) associate with specific proteins in order to regulate their entire cycle, including mRNA localization, translation and degradation, among others. The best characterized of such RNA-protein complexes, today named membraneless organelles, are Stress Granules (SGs) and Processing Bodies (PBs) which are involved in RNA storage and RNA decay/storage, respectively. Given that SGs and PBs are generally associated with repression of gene expression, viruses have evolved different mechanisms to counteract their assembly or to use them in their favor to successfully replicate within the host environment. In this review we summarize the current knowledge about the viral regulation of SGs and PBs, which could be a potential novel target for the development of broad-spectrum antiviral therapies.

Circadian rhythms are biological variables that oscillate with periods close to 24 h that are generated internally by biological clocks. Depending on the tissue/cell type, about 5–20% of genes are expressed rhythmically. Unexpectedly, the correlation between the oscillations of messengers and the proteins they encode is low. We hypothesize that these discrepancies could be because in certain phases of the circadian cycle some messengers could be translationally silenced and stored. Processing bodies (PBs) are membraneless organelles formed by ribonucleoprotein aggregates located in the cytoplasm. They contain silenced messengers and factors involved in mRNA processing. A previous work showed that the number of cells containing these mRNA granules varies when comparing two time-points in U2OS cell cultures and that these differences disappear when an essential clock gene is silenced. Here we evaluate whether PBs oscillate in Neuro2A cells. We analyzed in cell cultures synchronized with dexamethasone the variations in the number, the signal intensity of the markers used (GE-1/HEDLS and DDX6), and the area of PBs between 8 and 68 h post-synchronization. All three parameters oscillated with periods compatible with a circadian regulated process. The most robust rhythm was the number of PBs. These rhythms could be generated by oscillations in proteins that have been involved in the nucleation of these foci such as LSM1, TTP, and BRF1. The described phenomenon would allow to explain the differences observed in the temporal profiles of some messengers and their proteins and to understand how circadian clocks can control post-transcriptionally cellular functions.

Circadian rhythms are biological variables that oscillate with periods close to 24 h that are generated internally by biological clocks. Depending on the tissue/cell type, about 5–20% of genes are expressed rhythmically. Unexpectedly, the correlation between the oscillations of messengers and the proteins they encode is low. We hypothesize that these discrepancies could be because in certain phases of the circadian cycle some messengers could be translationally silenced and stored. Processing bodies (PBs) are membraneless organelles formed by ribonucleoprotein aggregates located in the cytoplasm. They contain silenced messengers and factors involved in mRNA processing. A previous work showed that the number of cells containing these mRNA granules varies when comparing two time-points in U2OS cell cultures and that these differences disappear when an essential clock gene is silenced. Here we evaluate whether PBs oscillate in Neuro2A cells. We analyzed in cell cultures synchronized with dexamethasone the variations in the number, the signal intensity of the markers used (GE-1/HEDLS and DDX6), and the area of PBs between 8 and 68 h post-synchronization. All three parameters oscillated with periods compatible with a circadian regulated process. The most robust rhythm was the number of PBs. These rhythms could be generated by oscillations in proteins that have been involved in the nucleation of these foci such as LSM1, TTP, and BRF1. The described phenomenon would allow to explain the differences observed in the temporal profiles of some messengers and their proteins and to understand how circadian clocks can control post-transcriptionally cellular functions. [ABSTRACT FROM AUTHOR]

Regulation of RNA homeostasis or "RNAstasis" is a central step in eukaryotic gene expression. From transcription to decay, cellular messenger RNAs (mRNAs) associate with specific proteins in order to regulate their entire cycle, including mRNA localization, translation and degradation, among others. The best characterized of such RNA-protein complexes, today named membraneless organelles, are Stress Granules (SGs) and Processing Bodies (PBs) which are involved in RNA storage and RNA decay/storage, respectively. Given that SGs and PBs are generally associated with repression of gene expression, viruses have evolved different mechanisms to counteract their assembly or to use them in their favor to successfully replicate within the host environment. In this review we summarize the current knowledge about the viral regulation of SGs and PBs, which could be a potential novel target for the development of broad-spectrum antiviral therapies. [ABSTRACT FROM AUTHOR]

Germ granules are hallmarks of all germ cells. Early ultrastructural studies in Drosophila first described these membraneless granules in the oocyte and early embryo as filled with amorphous to fibrillar material mixed with RNA. Genetic studies identified key protein components and specific mRNAs that regulate germ cell‐specific functions. More recently these ultrastructural studies have been complemented by biophysical analysis describing germ granules as phase‐transitioned condensates. In this review, we provide an overview that connects the composition of germ granules with their function in controlling germ cell specification, formation and migration, and illuminate these mysterious condensates as the gatekeepers of the next generation. [ABSTRACT FROM AUTHOR]

Activity-dependent translation requires the transport of mRNAs within membraneless protein assemblies known as neuronal granules from the cell body toward synaptic regions. Translation of mRNA is inhibited in these granules during transport but quickly activated in response to neuronal stimuli at the synapse. This raises an important question: how does synaptic activity trigger translation of oncesilenced mRNAs? Here, we demonstrate a strong connection between phase separation, the process underlying the formation of many different types of cellular granules, and in vitro inhibition of translation. By using the Fragile X Mental Retardation Protein (FMRP), an abundant neuronal granule component and translational repressor, we show that FMRP phase separates in vitro with RNA into liquid droplets mediated by its C-terminal low-complexity disordered region (i.e., FMRPLCR). FMRPLCR posttranslational modifications by phosphorylation and methylation have opposing effects on in vitro translational regulation, which corroborates well with their critical concentrations for phase separation. Our results, combined with bioinformatics evidence, are supportive of phase separation as a general mechanism controlling activity-dependent translation. [ABSTRACT FROM AUTHOR]

Abstract Cellular mRNAs cycle between translating and non-translating pools, polysomes compose the translating pool, while RNA granules contain translationally-silenced mRNAs, where the RNAs are either stored in stress granules, or accumulate in processing bodies (PBs) or GW-bodies, which have an important role in RNA degradation. Viruses have developed measures to prevent the deleterious effects of these structures during their replication. Rotavirus, the most common agent of viral gastroenteritis, is capable of establishing a successful infection by counteracting several of the antiviral responses of its host. Here, we describe that in rotavirus-infected cells the distribution of several RNA binding proteins is changed causing the disaggregation of PBs, the relocalization of GW-body proteins, and the cytoplasmic accumulation of HuR, a predominantly nuclear protein. We show that this redistribution of proteins is more likely caused by the accumulation of viral RNA in the cytoplasm of infected-cells, where it might be acting as an RBP sponge. [ABSTRACT FROM AUTHOR]

RNA granules are ubiquitous. Composed of RNA-binding proteins and RNAs, they provide functional compartmentalization within cells. They are inextricably linked with RNA biology and as such are often referred to as the hubs for post-transcriptional regulation. Much of the attention has been given to the proteins that form these condensates and thus many fundamental questions about the biology of RNA granules remain poorly understood: How and which RNAs enrich in RNA granules, how are transcripts regulated in them, and how do granule-enriched mRNAs shape the biology of a cell? In this review, we discuss the imaging, genetic, and biochemical data, which have revealed that some aspects of the RNA biology within granules are carried out by the RNA itself rather than the granule proteins. Interestingly, the RNA structure has emerged as an important feature in the post-transcriptional control of granule transcripts. This review is part of the Special Issue in the Frontiers in RNA structure in the journal Molecules.

RNA granules, aggresomes, and autophagy are key players in the immune response to viral infections. They provide countermeasures that regulate translation and proteostasis in order to rewire cell signaling, prevent viral interference, and maintain cellular homeostasis. The formation of cellular aggregates and inclusions is one of the strategies to minimize viral infections and virus-induced cell damage and to promote cellular survival. However, viruses have developed several strategies to interfere with these cellular processes in order to achieve productive replication within the host cells. A review on how these mechanisms could function as modulators of cell signaling and antiviral factors will be instrumental in refining the current scientific knowledge and proposing means whereby cellular granules and aggregates could be induced or prevented to enhance the antiviral immune response in mammalian cells.

DDX6 helicase is an RNA-binding protein involved in different aspects of gene expression regulation. The roles played by DDX6 depend on the complexes associated with it. Here, for the first time, we characterize the protein complexes associated with DDX6 in human adipose tissue-derived stem cells (hASCs) and analyze the dynamics of this helicase under different conditions of translational activity and differentiation. The results obtained demonstrated that the DDX6 helicase is associated with proteins involved in the control of mRNA localization, translation and metabolism in hASCs. DDX6 complexes may also assemble into more complex structures, such as RNA-dependent granules, the abundance and composition of which change upon inhibited translational activity. This finding supports the supposition that DDX6 is possibly involved in the regulation of the mRNA life cycle in hASCs. Although there was no significant variation in the protein composition of these complexes during early adipogenic or osteogenic induction, there was a change in the distribution pattern of DDX6: the number of DDX6 granules per cell was reduced during adipogenesis and was enhanced during osteogenesis.

Spatiotemporal translational regulation plays a key role in determining cell fate and function. Specifically, in neurons, local translation in dendrites is essential for synaptic plasticity and long-term memory formation. To achieve local translation, RNA-binding proteins in RNA granules regulate target mRNA stability, localization, and translation. To date, mRNAs localized to dendrites have been identified by comprehensive analyses. In addition, mRNAs associated with and regulated by RNA-binding proteins have been identified using various methods in many studies. However, the results obtained from these numerous studies have not been compiled together. In this review, we have catalogued mRNAs that are localized to dendrites and are associated with and regulated by the RNA-binding proteins fragile X mental retardation protein (FMRP), RNA granule protein 105 (RNG105, also known as Caprin1), Ras-GAP SH3 domain binding protein (G3BP), cytoplasmic polyadenylation element binding protein 1 (CPEB1), and staufen double-stranded RNA binding proteins 1 and 2 (Stau1 and Stau2) in RNA granules. This review provides comprehensive information on dendritic mRNAs, the neuronal functions of mRNA-encoded proteins, the association of dendritic mRNAs with RNA-binding proteins in RNA granules, and the effects of RNA-binding proteins on mRNA regulation. These findings provide insights into the mechanistic basis of protein-synthesis-dependent synaptic plasticity and memory formation and contribute to future efforts to understand the physiological implications of local regulation of dendritic mRNAs in neurons.

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.

Autosomal-dominant mutations within the gene FUS (fused in sarcoma) are responsible for 5% of familial cases of amyotrophic lateral sclerosis (ALS). The FUS protein is physiologically mainly located in the nucleus, while cytoplasmic FUS aggregates are pathological hallmarks of FUS-ALS. Data from non-neuronal cell models and/or models using heterologous expression of FUS mutants suggest cytoplasmic FUS translocation as a pivotal initial event which leads to neurodegeneration depending on a second hit. Here we present the first human model of FUS-ALS using patient-derived neurons carrying endogenous FUS mutations leading to a benign (R521C) or a more severe clinical phenotype (frameshift mutation R495QfsX527). We thereby showed that the severity of the underlying FUS mutation determines the amount of cytoplasmic FUS accumulation and cellular vulnerability to exogenous stress. Cytoplasmic FUS inclusions formed spontaneously depending on both, severity of FUS mutation and neuronal aging. These aggregates showed typical characteristics of FUS-ALS including methylated FUS. Finally, neurodegeneration was not specific to layer V cortical neurons perfectly in line with the current model of disease spreading in ALS. Our study highlights the value and usefulness of patient-derived cell models in FUS-ALS.

The fragile X messenger ribonucleoprotein 1 (FMRP) is a multifunctional RNA-binding protein implicated in human neurodevelopmental and neurodegenerative disorders. FMRP mediates the localization and activity-dependent translation of its associated mRNAs through the formation of phase-separated condensates that are trafficked by microtubule-based motors in axons. Axonal transport and localized mRNA translation are critical processes for long-term neuronal survival and are closely linked to the pathogenesis of neurological diseases. FMRP dynein-mediated axonal trafficking is still largely unexplored but likely to constitute a key process underlying FMRP spatiotemporal translational regulation. Here, we show that dynein light chain roadblock 1 (Dynlrb1), a subunit of the dynein complex, is a critical regulator of FMRP function. In sensory axons, FMRP associates with endolysosomal organelles, likely through annexin A11, and is retrogradely trafficked by the dynein complex in a Dynlrb1-dependent manner. Moreover, Dynlrb1 silencing induced FMRP granule accumulation and repressed the translation of microtubule-associated protein 1b, one of its primary mRNA targets. Our findings suggest that Dynlrb1 regulates FMRP function through the control of its transport and targeted degradation., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract P granules are RNA/protein condensates in the germline of Caenorhabditis elegans. Genetic analyses have begun to identify the proteins that regulate P granule assembly in the cytoplasm of zygotes. Among them, the RGG-domain protein PGL-3, the intrinsically disordered protein MEG-3, and the RNA helicase LAF-1 all bind and phase separate with RNA in vitro. We discuss how RNA-induced phase separation, competition with other RNA-binding proteins, and reversible phosphorylation contribute to the asymmetric localization of P granules in the cytoplasm of newly fertilized embryos. P granules contain RNA silencing complexes that monitor the germline transcriptome and may provide an RNA memory of germline gene expression across generations. Graphical Abstract Unlabelled Image Highlights • P granules are non-homogeneous, multi-phase condensates in the germline of C. elegans. • Proteins required for P granule assembly form phase-separated condensates with RNA in vitro. • Competition for RNA and reversible phosphorylation regulates the assembly and disassembly of P granules in embryos. • In developing gametes, P granules associate with nuclear pores and prevent the accumulation of non-germline transcripts. [ABSTRACT FROM AUTHOR]

Abstract: Over the last few years, many reports have defined several types of RNA cell granules composed of proteins and messenger RNA (mRNA) that regulate gene expression on a post‐transcriptional level. Processing bodies (P‐bodies) and stress granules (SGs) are among the best‐known RNA granules, only detectable when they accumulate into very dynamic cytosolic foci. Recently, a tight association has been found between positive‐stranded RNA viruses, including hepatitis C virus (HCV), and these granules. The present article offers a comprehensive review on the complex and paradoxical relationship between HCV, P‐bodies and SGs from a translational perspective. Despite the fact that components of P‐bodies and SGs have assiduously controlled mRNA expression, either by sequestration or degradation, for thousands of years, HCV has learned how to dangerously exploit certain of them for its own benefit in an endless biological war. Thus, HCV has gained the ability to hack ancient host machineries inherited from prokaryotic times. While P‐bodies and SGs are crucial to the HCV cycle, in the interferon‐free era we still lack detailed knowledge of the mechanisms involved, processes that may underlie the long‐term complications of HCV infection. [ABSTRACT FROM AUTHOR]

RNA granules are membraneless condensates that provide functional compartmentalization within cells. The mechanisms by which RNA granules form are under intense investigation. Here, we characterize the role of mRNAs and proteins in the formation of germ granules in Drosophila. Super-resolution microscopy reveals that the number, size, and distribution of germ granules is precisely controlled. Surprisingly, germ granule mRNAs are not required for the nucleation or the persistence of germ granules but instead control their size and composition. Using an RNAi screen, we determine that RNA regulators, helicases, and mitochondrial proteins regulate germ granule number and size, while the proteins of the endoplasmic reticulum, nuclear pore complex, and cytoskeleton control their distribution. Therefore, the protein-driven formation of Drosophila germ granules is mechanistically distinct from the RNA-dependent condensation observed for other RNA granules such as stress granules and P-bodies. [Display omitted] • The number, size, and distribution of germ granules is precisely regulated • Oskar germ granules do not require RNA for their nucleation and stability • RNA regulators and mitochondrial proteins control germ granule number and size • ER, nuclear pore, and cytoskeletal proteins regulate germ granule distribution Curnutte et al. observe that germ granule proteins rather than mRNAs promote the nucleation and stability of Oskar germ granules in Drosophila. While RNA regulators and mitochondrial proteins control the number and size of germ granules, the proteins of the ER, nuclear pores, and cytoskeleton control their distribution. [ABSTRACT FROM AUTHOR]

RNA molecules cause the proteins involved in the formation of germ granules to coalesce into liquid droplets.

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.

In polarized cells, such as neurons, the synthesis of an mRNA does not ensure its proper cellular expression. Most mature transcripts require the association with RNA-binding proteins, resulting in the formation of RNA granules, which are then transported within the cytoplasm along the cytoskeleton and delivered to their proper subcellular locations, where they can be locally translated. Here we review current microscopy methods that have been developed to visualize RNA granule formation, transport and translation at the single cell level with a special emphasis on the MS2 and SunTag systems. They include the labeling of mRNAs and RNA-binding proteins in living cells or even the detection of newly synthesized proteins in situ . [ABSTRACT FROM AUTHOR]

Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca 2+ -regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure. Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3'UTRs as well as c-myc 5'UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the K D of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3'UTR with K D s in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5'UTR indicating some selectivity. Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca 2+ . The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Patil, Panchal, Røstbø, Romanyuk, Hollås, Brenk, Grindheim and Vedeler.)