Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly.

Exon junction complexes are deposited at exon-exon junctions during splicing. They are primarily known to activate non-sense mediated degradation of transcripts harbouring premature stop codons before the last intron. According to a popular model, exon-junction complexes accompany mRNAs to the cytoplasm where the first translating ribosome pushes them out. However, they are also removed by uncharacterized, translation-independent mechanisms. Little is known about kinetic and transcript specificity of these processes. Here we tag core subunits of exon-junction complexes with complementary split nanoluciferase fragments to obtain sensitive and quantitative assays for complex formation. Unexpectedly, exon-junction complexes form large stable mRNPs containing stalled ribosomes. Complex assembly and disassembly rates are determined after an arrest in transcription and/or translation. 85% of newly deposited exon-junction complexes are disassembled by a translation-dependent mechanism. However as this process is much faster than the translation-independent one, only 30% of the exon-junction complexes present in cells at steady state require translation for disassembly. Deep RNA sequencing shows a bias of exon-junction complex bound transcripts towards microtubule and centrosome coding ones and demonstrate that the lifetimes of exon-junction complexes are transcript-specific. This study provides a dynamic vision of exon-junction complexes and uncovers their unexpected stable association with ribosomes.

Small extracellular vesicles (sEVs)-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing.

Small extracellular vesicles (sEVs) are naturally occurring vesicles that have the potential to be manipulated to become promising drug delivery vehicles for on-demand in vitro and in vivo gene editing. Here, we developed the modular safeEXO platform, a prototype sEV delivery vehicle that is mostly devoid of endogenous RNA and can efficaciously deliver RNA and ribonucleoprotein (RNP) complexes to their intended intracellular targets manifested by downstream biologic activity. We also successfully engineered producer cells to produce safeEXO vehicles that contain endogenous Cas9 (safeEXO-CAS) to effectively deliver efficient ribonucleoprotein (RNP)-mediated CRISPR genome editing machinery to organs or diseased cells in vitro and in vivo. We confirmed that safeEXO-CAS sEVs could co-deliver ssDNA, sgRNA and siRNA, and efficaciously mediate gene insertion in a dose-dependent manner. We demonstrated the potential to target safeEXO-CAS sEVs by engineering sEVs to express a tissue-specific moiety, integrin alpha-6 (safeEXO-CAS-ITGA6), which increased their uptake to lung epithelial cells in vitro and in vivo. We tested the ability of safeEXO-CAS-ITGA6 loaded with EMX1 sgRNAs to induce lung-targeted editing in mice, which demonstrated significant gene editing in the lungs with no signs of morbidity or detectable changes in immune cell populations. Our results demonstrate that our modular safeEXO platform represents a targetable, safe, and efficacious vehicle to deliver nucleic acid-based therapeutics that successfully reach their intracellular targets. Furthermore, safeEXO producer cells can be genetically manipulated to produce safeEXO vehicles containing CRISPR machinery for more efficient RNP-mediated genome editing. This platform has the potential to improve current therapies and increase the landscape of treatment for various human diseases using RNAi and CRISPR approaches.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Fragile X Messenger Ribonucleoprotein Protein and Its Multifunctionality: From Cytosol to Nucleolus and Back.

Silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene and a consequent lack of FMR protein (FMRP) synthesis are associated with fragile X syndrome, one of the most common inherited intellectual disabilities. FMRP is a multifunctional protein that is involved in many cellular functions in almost all subcellular compartments under both normal and cellular stress conditions in neuronal and non-neuronal cell types. This is achieved through its trafficking signals, nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS), as well as its RNA and protein binding domains, and it is modulated by various post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and methylation. This review summarizes the recent advances in understanding the interaction networks of FMRP with a special focus on FMRP stress-related functions, including stress granule formation, mitochondrion and endoplasmic reticulum plasticity, ribosome biogenesis, cell cycle control, and DNA damage response.

Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle.

mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.

Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.

Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.

Proteomic Profiling of Messenger Ribonucleoproteins in Mouse Tissues Based on Formaldehyde Cross-Linking.

Messenger ribonucleoprotein particles (mRNPs) are vital for tissue-specific gene expression via mediating posttranscriptional regulations. However, proteomic profiling of proteins in mRNPs, i.e., mRNA-associated proteins (mRAPs), has been challenging at the tissue level. Herein, we report the development of formaldehyde cross-linking-based mRNA-associated protein profiling (FAXRAP), a chemical strategy that enables the identification of mRAPs in both cultured cells and intact mouse organs. Applying FAXRAP, tissue-specific mRAPs were systematically profiled in the mouse liver, kidney, heart, and brain. Furthermore, brain mRAPs in Parkinson's disease (PD) mouse model were investigated, which revealed a global decrease of mRNP assembly in the brain of mice with PD. We envision that FAXRAP will facilitate uncovering the posttranscriptional regulation networks in various biological systems.

eIF4F is a thermo-sensing regulatory node in the translational heat shock response.

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

Near-Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR-Cas9 Ribonucleoprotein for Brain Gene Editing.

The CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene-editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near-infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene-editing potential of the formulation was demonstrated in vitro at the single-cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.

Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods.

Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.

Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP).

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

TRIM21 is critical in regulating hepatocellular carcinoma growth and response to therapy by altering the MST1/YAP pathway.

Liver cancer is the sixth most common cancer and the third leading cause of cancer-related death globally. Despite efforts being made in last two decades in cancer diagnosis and treatment, the 5-year survival rate of liver cancer remains extremely low. TRIM21 participates in cancer metabolism, glycolysis, immunity, chemosensitivity and metastasis by targeting various substrates for ubiquitination. TRIM21 serves as a prognosis marker for human hepatocellular carcinoma (HCC), but the mechanism by which TRIM21 regulates HCC tumorigenesis and progression remains elusive. In this study, we demonstrated that TRIM21 protein levels were elevated in human HCC. Elevated TRIM21 expression was associated with HCC progression and poor survival. Knockdown of TRIM21 in HCC cell lines significantly impaired cell growth and metastasis and enhanced sorafenib-induced toxicity. Mechanistically, we found that knockdown of TRIM21 resulted in cytosolic translocation and inactivation of YAP. At the molecular level, we further identified that TRIM21 interacted and induced ubiquitination of MST1, which resulted in MST1 degradation and YAP activation. Knockdown of MST1 or overexpression of YAP reversed TRIM21 knockdown-induced impairment of HCC growth and chemosensitivity. Taken together, the current study demonstrates a novel mechanism that regulates the Hippo pathway and reveals TRM21 as a critical factor that promotes growth and chemoresistance in human HCC.

Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.

The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation. The first electroporation introduces ribonucleoproteins formed by Cas9D10A with two guide RNAs to target DNA, and the second introduces the same ribonucleoprotein complex to target DNA plus Cas13a with one guide RNA to target RNAs. For complete details on the use and execution of this protocol, please refer to Nix et al.1.

Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy.

Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.

Nuclear ribonucleoprotein RALY downregulates foot-and-mouth disease virus replication but antagonized by viral 3C protease.

The internal ribosome entry site (IRES) element constitutes a cis-acting RNA regulatory sequence that recruits the ribosomal initiation complex in a cap-independent manner, assisted by various RNA-binding proteins and IRES trans-acting factors. Foot-and-mouth disease virus (FMDV) contains a functional IRES element and takes advantage of this element to subvert host translation machinery. Our study identified a novel mechanism wherein RALY, a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family belonging to RNA-binding proteins, binds to the domain 3 of FMDV IRES via its RNA recognition motif residue. This interaction results in the downregulation of FMDV replication by inhibiting IRES-driven translation. Furthermore, our findings reveal that the inhibitory effect exerted by RALY on FMDV replication is not attributed to the FMDV IRES-mediated assembly of translation initiation complexes but rather to the impediment of 80S ribosome complex formation after binding with 40S ribosomes. Conversely, 3Cpro of FMDV counteracts RALY-mediated inhibition by the ubiquitin-proteasome pathway. Therefore, these results indicate that RALY, as a novel critical IRES-binding protein, inhibits FMDV replication by blocking the formation of 80S ribosome, providing a deeper understanding of how viruses recruit and manipulate host factors. IMPORTANCE: The translation of FMDV genomic RNA driven by IRES element is a crucial step for virus infections. Many host proteins are hijacked to regulate FMDV IRES-dependent translation, but the regulatory mechanism remains unknown. Here, we report for the first time that cellular RALY specifically interacts with the IRES of FMDV and negatively regulates viral replication by blocking 80S ribosome assembly on FMDV IRES. Conversely, RALY-mediated inhibition is antagonized by the viral 3C protease by the ubiquitin-proteasome pathway. These results would facilitate further understanding of virus-host interactions and translational control during viral infection.

Trim21 deficiency in mice increases HCC carcinogenesis in a NASH context and is associated with immune checkpoint upregulation.

The global pandemic of metabolic diseases has increased the incidence of hepatocellular carcinoma (HCC) in the context of non-alcoholic steatohepatitis (NASH). The downregulation of the E3 ubiquitin ligase TRIM21 has been linked to poor prognosis in different cancers including HCC. In order to investigate the role of TRIM21 in liver cancer progression on NASH, Trim21+/+ and Trim21-/- male mice were injected with streptozotocin at the neonatal stage. The hypoinsulinemic mice were then fed with a high-fat high-cholesterol diet (HFHCD) for 4, 8 or 12 weeks. All mice developed NASH which systematically resulted in HCC progression. Interestingly, compared to the Trim21+/+ control mice, liver damage was worsened in Trim21-/- mice, with more HCC nodules found after 12 weeks on HFHCD. Immune population analysis in the spleen and liver revealed a higher proportion of CD4+PD-1+ and CD8+PD-1+ T cells in Trim21-/- mice. The liver and HCC tumors of Trim21-/- mice also exhibited an increase in the number of PD-L1+ and CD68+ PD-L1+ cells. Thus, TRIM21 limits the emergence of HCC nodules in mice with NASH by potentially restricting the expression of PD-1 in lymphocytes and PD-L1 in tumors.

High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.

Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.

An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).

Xist ribonucleoproteins promote female sex-biased autoimmunity.

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.