PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5\ub4-splice-site selection causing tissue-specific defects

\ua9 The Author(s) 2024.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

Chikungunya virus requires cellular chloride channels for efficient genome replication

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes—causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In this study, we investigated the role of host-cell chloride (Cl-) channels on CHIKV replication.We demonstrate that specific pharmacological Cl- channel inhibitors significantly inhibit CHIKV replication in a dose-dependent manner, suggesting that Cl-channels are pro-viral factors in human cells. Further analysis of the effect of the inhibitors on CHIKV attachment, entry, viral protein expression and replicon replication demonstrated that Cl- channels are specifically required for efficient CHIKV genome replication. This was conserved in mosquito cells, where CHIKV replication and genome copy number was significantly reduced following Cl- channel inhibition. siRNA silencing identified chloride intracellular channels 1 and 4 (CLIC1 and CLIC4, respectively) as required for efficient CHIKV replication and protein affinity chromatography showed low levels of CLIC1 in complex with CHIKV nsP3, an essential component of the viral replication machinery. In summary, for the first time we demonstrate that efficient replication of the CHIKV genome depends on cellular Cl- channels, in both human and mosquito cells and identifies CLIC1 and CLIC4 as agonists of CHIKV replication in human cells. We observe a modest interaction, either direct or indirect, between CLIC1 and nsP3 and hypothesize that CLIC1 may play a role in the formation/maintenance of CHIKV replication complexes. These findings advance our molecular understanding of CHIKV replication and identify potential druggable targets for the treatment and prevention of CHIKV mediated disease

Glycogen myophosphorylase loss causes increased dependence on glucose in iPSC-derived retinal pigment epithelium

Loss of glycogen myophosphorylase (PYGM) expression results in an inability to break down muscle glycogen, leading to McArdle disease—an autosomal recessive metabolic disorder characterized by exercise intolerance and muscle cramps. While previously considered relatively benign, this condition has recently been associated with pattern dystrophy in the retina, accompanied by variable sight impairment, secondary to retinal pigment epithelial (RPE) cell involvement. However, the pathomechanism of this condition remains unclear. In this study, we generated a PYGM-null induced pluripotent stem cell (iPSC) line, and differentiated it into mature RPE to examine structural and functional defects, along with metabolite release into apical and basal media. Mutant RPE exhibited normal photoreceptor outer segment phagocytosis but displayed elevated glycogen levels, reduced transepithelial resistance, and increased cytokine secretion across the epithelial layer compared to isogenic wildtype controls. Additionally, decreased expression of the visual cycle component, RDH11, encoding 11-cis-retinol dehydrogenase, was observed in PYGM-null RPE. While glycolytic flux and oxidative phosphorylation levels in PYGM-null RPE were near normal, the basal oxygen consumption rate (OCR) was increased. OCR in response to physiological levels of lactate was significantly greater in wildtype compared to PYGM-null RPE. Inefficient lactate utilization by mutant RPE resulted in higher glucose dependence and increased glucose uptake from the apical medium in the presence of lactate, suggesting a reduced capacity to spare glucose for photoreceptor use. Metabolic tracing confirmed slower 13C-lactate utilization by PYGM-null RPE. These findings have key implications for retinal health since they likely underlie the vision impairment in individuals with McArdle disease

Ocena wpływu szczepów Trichoderma sp. Na stan sanitarny gleby oraz plonowanie kapusty pekińskiej

We conducted a two-year study to prove the antagonistic properties between three Trichoderma sp. strains entered into soil in compost carriers and plant pathogens of the Fusarium and Alternaria genera. Another aim of the study was to analyse the influence of these strains on the value of the biological fertility index (BIF) and the commercial yield of napa cabbage. Eleven combinations were used in the experiment, including a control variant, a variant with soil treated with a mineral fertiliser, one with manure and eight variants treated with tomato or onion waste composts. Some of them were inoculated with Trichoderma atroviride (T1) and/or T. harzianum (T2 and T3) isolates. Soil samples were collected at three terms of the investigations and they were subject to microbiological (the total count of moulds, Trichoderma sp., Fusarium sp. and Alternaria sp.) and enzymatic analyses (dehydrogenase and catalase activity). After harvesting the dry and fresh weight of napa cabbage leaves was measured. The total count of moulds and the commercial yield of napa cabbage were most strongly modified in the variant with the tomato waste compost inoculated with the T1 strain. The variants with the onion waste composts caused the greatest increase in the biological fertility index (BIF). The research showed that the phytosanitary properties of Trichoderma sp. resulted from the specificity of the species. The smallest count of Fusarium sp. was observed in the soil treated with the onion waste compost inoculated with the T1 strain. The smallest count of Alternaria sp. was noted after treatment with the tomato waste compost, which was simultaneously inoculated with two isolates, i.e. T1 and T2. Apart from that, the research proved that both types of composts were good carriers for Trichoderma sp. isolates. The tomato waste composts caused more intense proliferation of Trichoderma sp. in soil.Przeprowadzono dwuletnie badania, których celem było wykazanie właściwości antagonistycznych trzech szczepów Trichoderma sp. wprowadzonych do gleby na nośnikach w postaci kompostów, w stosunku do patogenów roślinnych z rodzaju Fusarium i Alternaria. Ponadto celem badań była analiza wpływu wyżej wymienionych szczepów na wartość wskaźnika żyzno- ści gleby (BIF) oraz plon handlowy kapusty pekińskiej. W doświadczeniu zastosowano jedenaście kombinacji, na które składał się obiekt kontrolny, gleba nawożona mineralnie, obornikiem oraz osiem obiektów, do których wprowadzono komposty wytworzone z odpadów pomidorowych lub cebulowych. Część z nich zainokulowano izolatami Trichoderma atroviride (T1) i/lub T. harzianum (T2 i T3). W trzech terminach badań pobierano próbki gleby, a następnie poddano je analizom mikrobiologicznym (ogólna liczba grzybów pleśniowych, Trichoderma sp., Fusarium sp., Alternaria sp.) oraz enzymatycznym (aktywność dehydrogenaz oraz katalazy). Ponadto po zbiorach roślin określono suchą i świeżą masę liści kapusty pekiń- skiej. Ogólna liczba grzybów pleśniowych oraz plon handlowy roślin w największym stopniu modyfikowane były w obiekcie z dodatkiem kompostu pomidorowego, zainokulowanego szczepem T1. Do wzrostu wartości biologicznego wskaźnika żyzności gleby (BIF) w największym stopniu przyczyniły się warianty z dodatkiem kompostów cebulowych. Stwierdzono, że wła- ściwości fitosanitarne Trichoderma sp. wynikały ze specyfiki gatunku i tak najniższą liczebność Fusarium sp. w glebie zaobserwowano po zastosowaniu szczepu T1 wprowadzonego na nośniku w postaci kompostu cebulowego, natomiast Alternaria sp. po wprowadzeniu do gleby kompostu pomidorowego, zainokulowanego jednocześnie dwoma izolatami T1 i T2. Ponadto wykazano, że obydwa rodzaje zastosowanych kompostów okazały się dobrym nośnikiem dla izolatów Trichoderma sp.. Silniejsze namnażanie się Trichoderma sp. w glebie spowodowane było jednakże dodatkiem kompostów wytworzonym z odpadów pomidorowych