Formation Of Ribonucleoprotein Granules Research Articles

RNP granules in ALS and neurodegeneration: From multifunctional membraneless organelles to therapeutic opportunities.

Amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases are characterised by dysfunction of a host of RNA-binding proteins (RBPs) and a severely disrupted RNA metabolism. Recently, RBP-harbouring phase-separated complexes, ribonucleoprotein (RNP) granules, have come into the limelight as "crucibles" of neuronal pathology in ALS. RNP granules are indispensable for the multitude of regulatory processes underlying cellular RNA metabolism and serve as critical organisers of cellular biochemistry. Neurons, highly specialised cells, heavily rely on RNP granules for efficient trafficking, signalling and stress responses. Multiple RNP granule components, primarily RBPs such as TDP-43 and FUS, are affected by ALS mutations. However, even in the absence of mutations, RBP proteinopathies represent pathophysiological hallmarks of ALS. Given the high local concentrations of RBPs and RNAs, their weakened or enhanced interactions within RNP granules disrupt their homeostasis. Thus, the physiological process of phase separation and RNP granule formation, vital for maintaining the high-functioning state of neuronal cells, becomes their Achilles heel. Here, we will review the recent literature on the causes and consequences of abnormal RNP granule functioning in ALS and related disorders. In particular, we will summarise the evidence for the network-level dysfunction of RNP granules in these conditions and discuss considerations for therapeutic interventions to target RBPs, RNP granules and their network as a whole.

Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy.

Mutations in RNA binding motif protein 20 (RBM20) are a common cause of familial dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine-rich (RS-rich) domain, which mediates nuclear localization. These mutations induce RBM20 mis-localization to form aberrant ribonucleoprotein (RNP) granules in the cytoplasm of cardiomyocytes and abnormal alternative splicing of cardiac genes, contributing to DCM. We used adenine base editing (ABE) and prime editing (PE) to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Using ABE to correct RBM20R634Q human iPSCs, we achieved 92% efficiency of A-to-G editing, which normalized alternative splicing of cardiac genes, restored nuclear localization of RBM20, and eliminated RNP granule formation. In addition, we developed a PE strategy to correct the RBM20R636S mutation in iPSCs and observed A-to-C editing at 40% efficiency. To evaluate the potential of ABE for DCM treatment, we also created Rbm20R636Q mutant mice. Homozygous (R636Q/R636Q) mice developed severe cardiac dysfunction, heart failure, and premature death. Systemic delivery of ABE components containing ABEmax-VRQR-SpCas9 and single-guide RNA by adeno-associated virus serotype 9 in these mice restored cardiac function as assessed by echocardiography and extended life span. As seen by RNA sequencing analysis, ABE correction rescued the cardiac transcriptional profile of treated R636Q/R636Q mice, compared to the abnormal gene expression seen in untreated mice. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.

Oxidative Stress and Oocyte Cryopreservation: Recent Advances in Mitigation Strategies Involving Antioxidants.

Oocyte cryopreservation is widely used in assisted-reproductive technology and animal production. However, cryopreservation not only induces a massive accumulation of reactive oxygen species (ROS) in oocytes, but also leads to oxidative-stress-inflicted damage to mitochondria and the endoplasmic reticulum. These stresses lead to damage to the spindle, DNA, proteins, and lipids, ultimately reducing the developmental potential of oocytes both in vitro and in vivo. Although oocytes can mitigate oxidative stress via intrinsic antioxidant systems, the formation of ribonucleoprotein granules, mitophagy, and the cryopreservation-inflicted oxidative damage cannot be completely eliminated. Therefore, exogenous antioxidants such as melatonin and resveratrol are widely used in oocyte cryopreservation to reduce oxidative damage through direct or indirect scavenging of ROS. In this review, we discuss analysis of various oxidative stresses induced by oocyte cryopreservation, the impact of antioxidants against oxidative damage, and their underlying mechanisms. We hope that this literature review can provide a reference for improving the efficiency of oocyte cryopreservation.

Abstract 11164: Precise Genomic Correction of RBM20 Mutations Rescues Dilated Cardiomyopathy

Introduction: Despite current medical advancements, effective treatment for familial cardiomyopathies remains challenging. Mutations in RNA binding motif protein 20 ( RBM20 ) are a common cause of dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within the arginine/serine rich (RS-rich) domain, which mediates nuclear localization. These mutations induce RBM20 mis-localization and abnormal alternative splicing of cardiac genes. Importantly, the formation of aberrant ribonucleoprotein (RNP) granules in the cytoplasm of cardiomyocytes may contribute to DCM. Hypothesis: Precise genomic editing using base editing (BE) or prime editing (PE) corrects the mutations and rescues the pathophysiology of RBM20-related DCM. Methods: We used adenine base editing (ABE) or PE to correct the pathogenic p.R634Q (c.1901 G>A) and p.R636S (c.1906 C>A) mutations in the RS-rich domain of RBM20 in human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Additionally, using a humanized Rbm20 mutant mouse (R636Q/R636Q), we assessed in vivo ABE correction for DCM treatment. Results: Correcting RBM20 R634Q human iPSC by ABE using ABEmax-VRQR-SpCas9 and sgRNA components, we showed 89% efficiency of A to G editing, recovery of alternative splicing of cardiac genes, restoration of nuclear localization of RBM20 and elimination of RNP granule formation. Additionally, we developed a PE strategy to correct the RBM20 R636S mutation in iPSCs and observed A to C editing at 40% efficiency. To evaluate the potential of ABE for DCM treatment, we systemically delivered ABE components by adeno-associated virus in postnatal Rbm20 mutant mice (R636Q/R636Q). Uncorrected R636Q/R636Q mice exhibited severe heart failure and premature death by 3-months of age. In contrast, R636Q/R636Q mice receiving systemic ABE components showed a substantial improvement of LV function and an increase in life span. As seen by RNA-seq analysis, ABE correction rescued the transcriptional profile of treated R636Q/R636Q mice, while untreated mice showed altered cardiac gene expression. Conclusions: Precise gene editing technologies provide an innovative approach to correct disease-causing genomic mutations in cardiovascular diseases.

Thermodynamics and kinetics of phase separation of protein-RNA mixtures by a minimal model

Intracellular liquid-liquid phase separation enables the formation of biomolecular condensates, such as ribonucleoprotein granules, which play a crucial role in the spatiotemporal organization of biomolecules (e.g., proteins and RNAs). Here, we introduce a patchy-particle polymer model to investigate liquid-liquid phase separation of protein-RNA mixtures. We demonstrate that at low to moderate concentrations, RNA enhances the stability of RNA-binding protein condensates because it increases the molecular connectivity of the condensed-liquid phase. Importantly, we find that RNA can also accelerate the nucleation stage of phase separation. Additionally, we assess how the capacity of RNA to increase the stability of condensates is modulated by the relative protein-protein/protein-RNA binding strengths. We find that phase separation and multiphase organization of multicomponent condensates is favored when the RNA binds with higher affinity to the lower-valency proteins in the mixture than to the cognate higher-valency proteins. Collectively, our results shed light on the roles of RNA in ribonucleoprotein granule formation and the internal structuring of stress granules.

Shared Cell Biological Functions May Underlie Pleiotropy of Molecular Interactions in the Germ Lines and Nervous Systems of Animals

Evolutionary developmental biology focuses on understanding the origin and evolution of extant biological variation, and the genetic basis for this variation. The genetic toolkit appears largely finite across animals, such that a combination of regulatory evolution, gene recruitment (co-option) and genetic modularity often allow morphological and developmental diversity to arise. Here we summarize a number of observations from across animals, which together suggest that many genes and gene product interaction “modules” originally characterized for their role in the germ line also have neural roles. We explore potential explanations for this observation, noting that in the context of the germ line, these genes appear to have molecular and biochemical properties that make them well-suited to breaking symmetry within cells. The resulting asymmetry is often caused by gene products co-localizing asymmetrically to sub-cellular, non-membrane-bound, electron dense compartments known as ribonucleoprotein (RNP) granules. RNP granules contain high concentrations of translationally quiescent messenger RNAs and proteins and are thought to act as hubs of localized translational control. We propose that the use of strict translational control, which may be achieved molecular processes important for RNP granule formation and/or small RNA-related processes, is an important property of and a commonality between the germ line and nervous tissues, and help explain, at least in part, the close relationship between these two tissue types.

Unwind and Relax: DDX3X RNA Helicase as a Critical Mediator of Cortical Neurogenesis

In this issue of Neuron, Lennox etal. (2020) report the largest cohort of patients to date with DDX3X syndrome, discovering unique genotype-phenotype relationships that inform molecular pathogenesis. They then uncover unique roles of DDX3X in cortical neuron development and ribonucleoprotein granule formation.

Stress granules regulate paraspeckles: RNP granule continuum at work.

Eukaryotic cells contain several types of RNA-protein membraneless macro-complexes – ribonucleoprotein (RNP) granules that form by liquid-liquid phase separation. These structures represent biochemical microreactors for a variety of cellular processes and also act as highly accurate sensors of changes in the cellular environment. RNP granules share multiple protein components, however, the connection between spatially separated granules remains surprisingly understudied. Paraspeckles are constitutive nuclear RNP granules whose numbers significantly increase in stressed cells. Our recent work using affinity-purified paraspeckles revealed that another type of RNP granule, cytoplasmic stress granule (SG), acts as an important regulator of stress-induced paraspeckle assembly. Our study demonstrates that despite their residency in different cellular compartments, the two RNP granules are closely connected. This study suggests that nuclear and cytoplasmic RNP granules are integral parts of the intracellular “RNP granule continuum” and that rapid exchange of protein components within this continuum is important for the temporal control of cellular stress responses. It also suggests that cells can tolerate and efficiently handle a certain level of phase separation, which is reflected in the existence of “bursts”, or “waves”, of RNP granule formation. Our study triggers a number of important questions related to the mechanisms controlling the flow of RNP granule components within the continuum and to the possibility of targeting these mechanisms in human disease.

Single-Molecule and Ensemble Methods to Probe Initial Stages of RNP Granule Assembly.

Ribonucleoprotein (RNP) granules are membraneless organelles, consisting of high local concentrations of RNA and proteins bearing intrinsically disordered regions (IDRs). They are formed by liquid-liquid phase separation (LLPS). In neurodegenerative diseases such as ALS, mutations in granule proteins such as FUS and TDP-43 accelerate abnormal liquid to solid transition of RNP granules, leading to formation of fiber-like structures. Methods to study granules must be carefully selected based on the stage of granule's life. Here we describe a strategic combination of single-molecule biophysical and ensemble biochemical techniques that may be employed to extract insightful information about early stages of RNP granule formation. Protein-RNA interaction and stoichiometry of the complex in the early soluble stage of RNP assembly can be probed by single-molecule FRET (smFRET) assay and electrophoretic mobility shift assay (EMSA), respectively. RNP-RNP interaction that likely contributes to RNP nucleation can be reported on by a smFRET-based RNA annealing assay. The next stage in the assembly pathway, that is, phase separation from diffused to liquid-like droplets, may be monitored by a phase separation assay. Finally, RNP granules isolated from mammalian cells can be investigated using a unique single-molecule pull-down (SiMPull) assay.

RNA Protein Granules Modulate tau Isoform Expression and Induce Neuronal Sprouting

The neuronal microtubule-associated protein Tau is expressed in different variants, and changes in Tau isoform composition occur during development and disease. Here, we investigate a potential role of the multivalent tau mRNA-binding proteins G3BP1 and IMP1 in regulating neuronal tau expression. We demonstrate that G3BP1 and IMP1 expression induces the formation of structures, which qualify as neuronal ribonucleoprotein (RNP) granules and concentrate multivalent proteins and mRNA. We show that RNP granule formation leads to a >30-fold increase in the ratio of high molecular weight to low molecular weight tau mRNA and an ∼12-fold increase in high molecular weight to low molecular weight Tau protein. We report that RNP granule formation is associated with increased neurite formation and enhanced process growth. G3BP1 deletion constructs that do not induce granule formation are also deficient in inducing neuronal sprouting or changing the expression pattern of tau. The data indicate that granule formation driven by multivalent proteins modulates tau isoform expression and suggest a morphoregulatory function of RNP granules during health and disease.

SCD6 induces ribonucleoprotein granule formation in trypanosomes in a translation-independent manner, regulated by its Lsm and RGG domains.

Ribonucleoprotein (RNP) granules are cytoplasmic, microscopically visible structures composed of RNA and protein with proposed functions in mRNA decay and storage. Trypanosomes have several types of RNP granules, but lack most of the granule core components identified in yeast and humans. The exception is SCD6/Rap55, which is essential for processing body (P-body) formation. In this study, we analyzed the role of trypanosome SCD6 in RNP granule formation. Upon overexpression, the majority of SCD6 aggregates to multiple granules enriched at the nuclear periphery that recruit both P-body and stress granule proteins, as well as mRNAs. Granule protein composition depends on granule distance to the nucleus. In contrast to findings in yeast and humans, granule formation does not correlate with translational repression and can also take place in the nucleus after nuclear targeting of SCD6. While the SCD6 Lsm domain alone is both necessary and sufficient for granule induction, the RGG motif determines granule type and number: the absence of an intact RGG motif results in the formation of fewer granules that resemble P-bodies. The differences in granule number remain after nuclear targeting, indicating translation-independent functions of the RGG domain. We propose that, in trypanosomes, a local increase in SCD6 concentration may be sufficient to induce granules by recruiting mRNA. Proteins that bind selectively to the RGG and/or Lsm domain of SCD6 could be responsible for regulating granule type and number.