Pre-mRNA Processing Factors and Retinitis Pigmentosa: RNA Splicing and Beyond

Retinitis pigmentosa (RP) is the most common inherited retinal disease characterized by progressive degeneration of photoreceptors and/or retinal pigment epithelium that eventually results in blindness. Mutations in pre-mRNA processing factors (PRPF3, 4, 6, 8, 31, SNRNP200, and RP9) have been linked to 15–20% of autosomal dominant RP (adRP) cases. Current evidence indicates that PRPF mutations cause retinal specific global spliceosome dysregulation, leading to mis-splicing of numerous genes that are involved in a variety of retina-specific functions and/or general biological processes, including phototransduction, retinol metabolism, photoreceptor disk morphogenesis, retinal cell polarity, ciliogenesis, cytoskeleton and tight junction organization, waste disposal, inflammation, and apoptosis. Importantly, additional PRPF functions beyond RNA splicing have been documented recently, suggesting a more complex mechanism underlying PRPF-RPs driven disease pathogenesis. The current review focuses on the key RP-PRPF genes, depicting the current understanding of their roles in RNA splicing, impact of their mutations on retinal cell’s transcriptome and phenome, discussed in the context of model species including yeast, zebrafish, and mice. Importantly, information on PRPF functions beyond RNA splicing are discussed, aiming at a holistic investigation of PRPF-RP pathogenesis. Finally, work performed in human patient-specific lab models and developing gene and cell-based replacement therapies for the treatment of PRPF-RPs are thoroughly discussed to allow the reader to get a deeper understanding of the disease mechanisms, which we believe will facilitate the establishment of novel and better therapeutic strategies for PRPF-RP patients

A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing

Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3′-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module. The composite double-stranded /single-stranded RNA-binding region assembles cooperatively with Snu13 and Prp31 on U4/U6 di-snRNAs and inhibits Brr2-mediated U4/U6 di-snRNA unwinding in vitro. RNP-disrupting mutations in Prp3 lead to U4/U6•U5 tri-snRNP assembly and splicing defects in vivo. Our results reveal how Prp3 acts as an important bridge between U4/U6 and U5 in the tri-snRNP and comparison with a Prp24-U6 snRNA recycling complex suggests how Prp3 may be involved in U4/U6 reassembly after splicing

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

Progressive accumulation of cytoplasmic aggregates in PRPF31 retinal pigment epithelium cells interferes with cell survival

Abstract Retinitis Pigmentosa (RP) is a common form of inherited degenerative disease that often leads to blindness. About 10% autosomal dominant RP cases have been associated with mutations in PRPF31 gene, which is involved in pre‐mRNA splicing. This commentary summarises the key findings of our recent publication ‘Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient‐induced pluripotent stem cell‐derived retinal pigment epithelium cells’ in the context of large cytoplasmic aggregates which accumulate progressive with time and impair cell function and survival. Understanding the pathomechanism of PRPF31‐RP provides invaluable information that can be used to understand other PRPF‐RPs, and help to design effective and appropriate therapeutic strategies for the treatment of RP patients with PRPF31 mutations

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

Insights Into the Structural Peculiarities of The\u3cem\u3e N\u3c/em\u3e-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

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

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