MERISTEM-DEFECTIVE regulates the balance between stemness and differentiation in the root meristem through RNA splicing control.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors.

Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation. Protein-protein crosslinking of NTC, functional assays in vitro, and assessment of its role in DNA damage response provide mechanistic insight into the organization of the NTC core and the communication between PLRG1 and Prp19 that enables E3 activity. This reveals a unique mode of regulation for a complex E3 ligase and advances understanding of its dynamics in various cellular pathways.

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes.

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.

The G-patch protein Spp2 couples the spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the spliceosome.

Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome. In the absence of Spp2, the B(act) spliceosome efficiently triggers Prp2's NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B(act) to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2's RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2's ATPase activity to remodeling of the spliceosome into a catalytically active machine.

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.

TWIST1 activates cancer stem cell marker genes to promote epithelial-mesenchymal transition and tumorigenesis in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. Overexpression of EMT master transcription factors can promote differentiated cells to undergo cancer reprogramming processes and acquire a stem cell-like status. METHODS: The KYSE-30 and YM-1 ESCC cell lines were transduced with retroviruses expressing TWIST1 or GFP and analyzed by quantitative reverse transcription PCR (qRT-PCR), chromatin immunoprecipitation (ChIP), and immunostaining to investigate the correlation between TWIST1 and stemness markers expression. Cells expressing TWIST1 were characterized for mRNA candidates by qRT-PCR and for protein candidates by Flow cytometry and Immunocytochemistry. TWIST1-ESCC cells were also evaluated for apoptosis and drug resistance. RESULTS: Here we identify a role for TWIST1 in the establishment of ESCC cancer stem cell (CSC)-like phenotype, facilitating the transformation of non-CSCs to CSCs. We provide evidence that TWIST1 expression correlates with the expression of CSC markers in ESCC cell lines. ChIP assay results demonstrated that TWIST1 regulates CSC markers, including CD44, SALL4, NANOG, MEIS1, GDF3, and SOX2, through binding to the E-box sequences in their promoters. TWIST1 promoted EMT through E-cadherin downregulation and vimentin upregulation. Moreover, TWIST1 expression repressed apoptosis in ESCC cells through upregulation of Bcl-2 and downregulation of the Bax protein, and increased ABCG2 and ABCC4 transporters expression, which may lead to drug resistance. CONCLUSIONS: These findings support a critical role for TWIST1 in CSC-like generation, EMT progression, and inhibition of apoptosis in ESCC. Thus, TWIST1 represents a therapeutic target for the suppression of esophageal cell transformation to CSCs and ESCC malignancy.

Hydroxyapatite as a biomaterial - a gift that keeps on giving.

The synthetic analogue to biogenic apatite, hydroxyapatite (HA) has a number of physicochemical properties that make it an attractive candidate for diagnosis, treatment of disease and augmentation of biological tissues. Here we describe some of the recent studies on HA, which may provide bases for a number of new medical applications. The content of this review is divided to different medical application modes utilizing HA, including tissue engineering, medical implants, controlled drug delivery, gene therapies, cancer therapies and bioimaging. A number of advantages of HA over other biomaterials emerge from this discourse, including (i) biocompatibility, (ii) bioactivity, (iii) relatively simple synthesis protocols for the fabrication of nanoparticles with specific sizes and shapes, (iv) smart response to environmental stimuli, (v) facile functionalization and surface modification through noncovalent interactions, and (vi) the capacity for being simultaneously loaded with a wide range of therapeutic agents and switched to bioimaging modalities for uses in theranostics. A special section is dedicated to analysis of the safety of particulate HA as a component of parenterally administrable medications. It is concluded that despite the fact that many benefits come with the usage of HA, its deficiencies and potential side effects must be addressed before the translation to the clinical domain is pursued. Although HA has been known in the biomaterials world as the exemplar of safety, this safety proves to be the function of size, morphology, surface ligands and other structural and compositional parameters defining the particles. For this reason, each HA, especially when it comes in a novel structural form, must be treated anew from the safety research angle before being allowed to enter the clinical stage.

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA.

The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 5' splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.

Novel regulatory principles of the spliceosomal Brr2 RNA helicase and links to retinal disease in humans.

For each round of pre-mRNA splicing, a spliceosome is assembled anew on its substrate. RNA-protein remodeling events required for spliceosome assembly, splicing catalysis, and spliceosome disassembly are driven and controlled by a conserved group of ATPases/RNA helicases. The activities of most of these enzymes are timed by their recruitment to the spliceosome. The Brr2 enzyme, however, which mediates spliceosome catalytic activation, is a stable subunit of the spliceosome, and thus, requires special regulation. Recent structural and functional studies have revealed diverse mechanisms whereby an RNaseH-like and a Jab1/MPN-like domain of the Prp8 protein regulate Brr2 activity during splicing both positively and negatively. Reversible Brr2 inhibition might in part be achieved via an intrinsically unstructured element of the Prp8 Jab1/MPN domain, a concept widespread in biological systems. Mutations leading to changes in the Prp8 Jab1/MPN domain, which are linked to a severe form of retinitis pigmentosa, disrupt Jab1/MPN-mediated regulation of Brr2.

The effect of modification of DNA interference on myostatin gene expression in mice.

Myostatin is a known negative regulator of muscle tissue growth. Thus, an inhibitor of myostatin may be therapeutically useful as an anabolic agent for the muscle tissue. A promising gene-silencing approach for gene therapy is DNA interference (DNAi), a sequence that is complementary to the promoter region of a target gene. To confer resistance to nuclease digestion, several modifications such as methylphosphonate or phosphorothioate have been proposed, wherein a nonbridging oxygen atom in the oligonucleotide phosphate backbone is replaced by sulphur. The aim of the present study was to assess the effectiveness of the DNAi molecule with phosphorothioate (PS) and without phosphorothioate (WPS) modification for inhibition of myostatin gene expression in mice. Eighteen four-week-old male BALB/c mice were randomly divided into three groups: DNAi-PS (n = 6), DNAi-WPS (n = 6) and control (n = 6). Intraperitoneal injections of DNAi (10 mg/kg) were given once a week, and mice body weights were measured weekly and sacrificed after three weeks. The expression of myostatin was assessed using real-time quantitative polymerace chain reaction. For histological evaluation, the skeletal muscle tissue was dissected from the biceps. The results were analysed by a t-test. Results demonstrated that administration of DNAi intraperitoneally with modification could suppress myostatin expression by up to 70%. Leg weight and histological analysis proved that chemically modified DNAi significantly suppressed the myostatin gene in mice. Overall, the results on DNA-induced gene silencing by antisense DNA oligonucleotides in animals can provide insight into the treatment of inherited diseases.

Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.

An Update on the Application of CRISPR Technology in Clinical Practice.

The CRISPR/Cas system, an innovative gene-editing tool, is emerging as a promising technique for genome modifications. This straightforward technique was created based on the prokaryotic adaptive immune defense mechanism and employed in the studies on human diseases that proved enormous therapeutic potential. A genetically unique patient mutation in the process of gene therapy can be corrected by the CRISPR method to treat diseases that traditional methods were unable to cure. However, introduction of CRISPR/Cas9 into the clinic will be challenging because we still need to improve the technology's effectiveness, precision, and applications. In this review, we first describe the function and applications of the CRISPR-Cas9 system. We next delineate how this technology could be utilized for gene therapy of various human disorders, including cancer and infectious diseases and highlight the promising examples in the field. Finally, we document current challenges and the potential solutions to overcome these obstacles for the effective use of CRISPR-Cas9 in clinical practice.

AI-driven covalent drug design strategies targeting main protease (mpro) against SARS-CoV-2: structural insights and molecular mechanisms.

The emergence of new SARS-CoV-2 variants has raised concerns about the effectiveness of COVID-19 vaccines. To address this challenge, small-molecule antivirals have been proposed as a crucial therapeutic option. Among potential targets for anti-COVID-19 therapy, the main protease (Mpro) of SARS-CoV-2 is important due to its essential role in the virus's life cycle and high conservation. The substrate-binding region of the core proteases of various coronaviruses, including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV), could be used for the generation of new protease inhibitors. Various drug discovery methods have employed a diverse range of strategies, targeting both monomeric and dimeric forms, including drug repurposing, integrating virtual screening with high-throughput screening (HTS), and structure-based drug design, each demonstrating varying levels of efficiency. Covalent inhibitors, such as Nirmatrelvir and MG-101, showcase robust and high-affinity binding to Mpro, exhibiting stable interactions confirmed by molecular docking studies. Development of effective antiviral drugs is imperative to address potential pandemic situations. This review explores recent advances in the search for Mpro inhibitors and the application of artificial intelligence (AI) in drug design. AI leverages vast datasets and advanced algorithms to streamline the design and identification of promising Mpro inhibitors. AI-driven drug discovery methods, including molecular docking, predictive modeling, and structure-based drug repurposing, are at the forefront of identifying potential candidates for effective antiviral therapy. In a time when COVID-19 potentially threat global health, the quest for potent antiviral solutions targeting Mpro could be critical for inhibiting the virus.Communicated by Ramaswamy H. Sarma.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

An overview of the vaccine platforms to combat COVID-19 with a focus on the subunit vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that has caused the recent coronavirus disease (COVID-19) global pandemic. The current approved COVID-19 vaccines have shown considerable efficiency against hospitalization and death. However, the continuation of the pandemic for more than two years and the likelihood of new strain emergence despite the global rollout of vaccination highlight the immediate need for the development and improvement of vaccines. mRNA, viral vector, and inactivated virus vaccine platforms were the first members of the worldwide approved vaccine list. Subunit vaccines. which are vaccines based on synthetic peptides or recombinant proteins, have been used in lower numbers and limited countries. The unavoidable advantages of this platform, including safety and precise immune targeting, make it a promising vaccine with wider global use in the near future. This review article summarizes the current knowledge on different vaccine platforms, focusing on the subunit vaccines and their clinical trial advancements against COVID-19.

Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.

Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.

Characterization of stability, safety and immunogenicity of the mRNA lipid nanoparticle vaccine Iribovax® against COVID-19 in nonhuman primates.

mRNA-lipid nanoparticle (mRNA-LNP) vaccines have proved their efficacy, versatility and unprecedented manufacturing speed during the COVID-19 pandemic. Here we report on the physicochemical properties, thermostability, immunogenicity, and protective efficacy of the nucleoside-modified mRNA-LNP vaccine candidate Iribovax® (also called SNEG2c). Injection of BALB/c mice, rabbits and nonhuman primates with two doses of SNEG2c induced production of high-titers of SARS-CoV-2 spike-specific and receptor-binding domain (RBD)-neutralizing antibodies in immunized animals. In addition to the strong humoral response, SNEG2c elicited substantial Th1-biased T-cell response. Sera from rhesus macaques immunized with a low dose of the vaccine showed robust spike-specific antibody titers 3-24× as high as those in convalescent sera from a panel of COVID-19 patients and 50% virus neutralization geometric mean titer of 1024 against SARS-CoV-2. Strikingly, immunization with SNEG2c completely cleared infectious SARS-CoV-2 from the upper and lower respiratory tracts of challenged macaques and protected them from viral-induced lung and trachea lesions. In contrast, the non-vaccinated macaques developed moderate to severe pulmonary pathology after the viral challenge. We present the results of repeat-dose and local tolerance toxicity and thermostability studies showing how the physicochemical properties of the mRNA-LNPs change over time and demonstrating that SNEG2 is safe, well tolerated and stable for long-term. These results support the planned human trials of SNEG2c.

Insights into the structural peculiarities of the N-terminal and receptor binding domains of the spike protein from the SARS-CoV-2 Omicron variant.

Since the new variant of SARS-CoV-2, Omicron (BA.1) has raised serious concerns, it is important to investigate the effects of mutations in the NTD and RBD domains of the spike protein for the development of COVID-19 vaccines. In this study, computational analysis of the Wuhan and Omicron NTDs and RBDs in their unbound and bound states to mAb 4A8 and ACE2 were performed. In addition, the interaction of NTD with antibody and RBD with ACE2 were evaluated in the presence of long glycans. The results show that long glycans at the surface of NTDs can reduce the accessibility of protein epitopes, thereby reducing binding efficiency and neutralizing potency of specific antibodies. Also, our findings indicate that the existence of the long glycans result in increased stability and enhanced affinity of the RBD to ACE2 in the Wuhan and Omicron variant. Key residues that play an important role in increasing the structural stability of the protein were identified using RIN analysis and in the state of interaction with mAb 4A8 and ACE2 through per-residue decomposition analysis. Further, the results of the free energy binding calculation using MM/GBSA method show that the Omicron variant has a higher infectivity than the Wuhan. This study provides a better understanding of the structural changes in the spike protein and can be useful for the development of novel therapeutics.

Developing novel liquid biopsy by selective capture of viral RNA on magnetic beads to detect COVID-19.

Objectives: Early, specific, and sensitive detection methods of COVID-19 are essential for force stopping its worldwide infection. Although CT images of the lung and/or viral RNA extraction followed by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) are widely used; they have some limitations. Here, we developed a highly sensitive magnetic bead-based viral RNA extraction assay followed by rRT-PCR. Materials and Methods: Case group included oropharyngeal/nasopharyngeal and blood samples from 30 patients diagnosed positive by PCR test for COVID-19 and control group included 30 same samples from COVID-19 negative PCR test individuals. RNA was extracted, using viral RNA extraction kit as well as using our hand-made capture bead-based technique. A one-step cDNA synthesis and Real Time PCR was conducted. A two-step comparison of the different viral RNA extraction methods for oropharyngeal/nasopharyngeal and blood samples was performed. Student t-test was applied with a P<0.05 considered statistically significant. Results: In the case group, all 30 mucosal samples extracted either with viral RNA extraction kit or with beads-based assay were COVID-19 positive although in the latter category, Cqs were much lower. Although 43% of plasma samples extracted by bead-based method were found to be positive but no plasma samples extracted with column-based kit were detected positive by Real Time PCR. Conclusion: Bead-based RNA extraction method can reduce RNA loss by its single-tube performance and enhance the test sensitivity. It is also more sensitive to lower viral loads as shown in the detection of blood samples and the lower Cqs of mucosal samples.

Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells.

INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.