Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.

Genetic information flows from DNA to protein through RNA in the central dogma. Different RNA species are known to accomplish essential tasks of protein encoding (mRNAs), amino acid loading (tRNAs), and translation machinery assembly (rRNAs). However, on top of these well-known roles, RNAs are central to various cellular regulatory pathways. Here we summarize newly emerging regulatory functions of RNA, specifically focusing on regulations through RNA modifications, RNP granules, and chromatin-associated regulatory RNA. In addition to being an essential building block of the central dogma, RNA can be critical to the regulation of many cellular processes.

Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity.

Ribonucleoprotein granules are bio-condensates that form a diverse group of dynamic membrane-less organelles implicated in several cellular functions, including stress response and cellular survival. In Toxoplasma gondii, a type of bio-condensates referred to as stress granules (SGs) are formed prior to the parasites\' egress from the host cell and are implicated in the survival and invasion competency of extracellular tachyzoites. We used paraformaldehyde to fix and cross-link SG proteins to allow purification by centrifugation and analysis by mass spectrometry. We profiled protein components of SGs at 10 and 30 min post-egress when parasite\'s invasion ability is significantly diminished. Thirty-three proteins were identified from 10 min SGs, and additional 43 proteins were identified from 30 min SGs. Notably, common SG components such as proteins with intrinsically disordered domains were not identified. Gene ontology analysis of both 10 and 30 min SGs shows that overall molecular functions of SGs\' proteins are ATP-binding, GTP-binding, and GTPase activity. Discernable differences between 10 and 30 min SGs are in the proportions of translation and microtubule-related proteins. Ten-minute SGs have a higher proportion of microtubule-related proteins and a lower proportion of ribosome-related proteins, while a reverse correlation was identified for those of 30 min. It remains to be investigated whether this reverse correlation contributes to the ability of extracellular tachyzoites to reinvade host cells.

Cellular biomolecular condensates, termed ribonucleoprotein (RNP) granules, are often enriched in messenger RNA (mRNA) molecules relative to the surrounding cytoplasm. Yet, the spatial localization and diffusion of mRNAs in close proximity to phase separated RNP granules are not well understood. In this study, we performed single-molecule fluorescence imaging experiments of mRNAs in live cells in the presence of two types of RNP granules, stress granules (SGs) and processing bodies (PBs), which are distinct in their molecular composition and function. We developed a photobleaching- and noise-corrected colocalization imaging algorithm that was employed to determine the accurate positions of individual mRNAs relative to the granule\'s boundaries. We found that mRNAs are often localized at granule boundaries, an observation consistent with recently published data. We suggest that mRNA molecules become spontaneously confined at the RNP granule boundary similar to the adsorption of polymer molecules at liquid-liquid interfaces, which is observed in various technological and biological processes. We also suggest that this confinement could be due to a combination of intermolecular interactions associated with, first, the screening of a portion of the RNP granule interface by the polymer and, second, electrostatic interactions due to a strong electric field induced by a Donnan potential generated across the thin interface.

Transposable elements (TEs) are DNA sequences that can transpose and replicate within the genome, leading to genetic changes that affect various aspects of host biology. Evolutionarily, hosts have also developed molecular mechanisms to suppress TEs at the transcriptional and post-transcriptional levels. Recent studies suggest that stress-induced formation of ribonucleoprotein (RNP) granules, including stress granule (SG) and processing body (P-body), can play a role in the sequestration of TEs to prevent transposition, suggesting an additional layer of the regulatory mechanism for TEs. RNP granules have been shown to contain factors involved in RNA regulation, including mRNA decay enzymes, RNA-binding proteins, and noncoding RNAs, which could potentially contribute to the regulation of TEs. Therefore, understanding the interplay between TEs and RNP granules is crucial for elucidating the mechanisms for maintaining genomic stability and controlling gene expression. In this review, we provide a brief overview of the current knowledge regarding the interplay between TEs and RNP granules, proposing RNP granules as a novel layer of the regulatory mechanism for TEs during stress.

Significance: Stress granules (SGs) are biomolecular condensates that form upon global translation suppression during stress. SGs are enriched in translation factors and messenger RNAs (mRNAs), which they may sequester away from the protein synthesis machinery. While this is hypothesized to remodel the functional transcriptome during stress, it remains unclear whether SGs are a cause, or simply a consequence, of translation repression. Understanding the function of SGs is particularly important because they are implicated in numerous diseases including viral infections, cancer, and neurodegeneration. Recent Advances: We synthesize recent SG research spanning biological scales, from observing single proteins and mRNAs within one cell to measurements of the entire transcriptome or proteome of SGs in a cell population. We use the emerging understanding from these studies to suggest that SGs likely have less impact on global translation, but instead may strongly influence the translation of individual mRNAs localized to them. Critical Issues: Development of a unified model that links stress-induced RNA-protein condensation to regulation of downstream gene expression holds promise for understanding the mechanisms of cellular resilience. Future Directions: Therefore, upcoming research should clarify what influence SGs exert on translation at all scales as well as the molecular mechanisms that enable this. The resulting knowledge will be required to drive discovery in how SGs allow organisms to adapt to challenges and support health or go awry and lead to disease. Antioxid. Redox Signal. 39, 390-409.

In animals with germ plasm, specification of the germline involves \'germ granules\', cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In Caenorhabditis elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here, we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, \'germline P-bodies\' contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, misregulate POS-1 targets, mis-specify the germline founder cell and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells. This article has an associated \'The people behind the papers\' interview.

Cells possess membraneless ribonucleoprotein (RNP) granules, including stress granules, processing bodies, Cajal bodies, or paraspeckles, that play physiological or pathological roles. RNP granules contain RNA and numerous RNA-binding proteins, transiently formed through the liquid-liquid phase separation. The assembly or disassembly of numerous RNP granules is strongly controlled to maintain their homeostasis and perform their cellular functions properly. Normal RNA granules are reversibly assembled, whereas abnormal RNP granules accumulate and associate with various neurodegenerative diseases. This review summarizes current studies on the physiological or pathological roles of post-translational modifications of various cellular RNP granules and discusses the therapeutic methods in curing diseases related to abnormal RNP granules by autophagy.

Dynamic and local adjustments of the axonal proteome are observed in response to extracellular cues and achieved via translation of axonally localized mRNAs. To be localized, these mRNAs must be recognized by RNA binding proteins and packaged into higher-order ribonucleoprotein (RNP) granules transported along axonal microtubules via molecular motors. Axonal recruitment of RNP granules is not constitutive, but rather regulated by external signals such as developmental cues, through pathways yet to be identified. The Drosophila brain represents an excellent model system where to study the transport of RNP granules as it is triggered in specific populations of neurons undergoing remodeling during metamorphosis. Here, we describe a protocol enabling live imaging of axonal RNP granule transport with high spatiotemporal resolution in Drosophila maturing brains. In this protocol, pupal brains expressing endogenous or ectopic fluorescent RNP components are dissected, mounted in a customized imaging chamber, and imaged with an inverted confocal microscope equipped with sensitive detectors. Axonal RNP granules are then individually tracked for further analysis of their trajectories. This protocol is rapid (less than 1 hour to prepare brains for imaging) and is easy to handle and to implement.