Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells
RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in ret...
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2025-07-01
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| author | Rebecca D. Miller Isaac Mondon Charles Ellis Anna-Marie Muir Stephanie Turner Eloise Keeling Htoo A. Wai David S. Chatelet David A. Johnson David A. Tumbarello Andrew J. Lotery Diana Baralle J. Arjuna Ratnayaka |
| author_facet | Rebecca D. Miller Isaac Mondon Charles Ellis Anna-Marie Muir Stephanie Turner Eloise Keeling Htoo A. Wai David S. Chatelet David A. Johnson David A. Tumbarello Andrew J. Lotery Diana Baralle J. Arjuna Ratnayaka |
| author_sort | Rebecca D. Miller |
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| description | RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. |
| format | Article |
| id | doaj-art-afdec3b48b364548bebf34cf94e9c6a6 |
| institution | Kabale University |
| issn | 2073-4409 |
| language | English |
| publishDate | 2025-07-01 |
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| series | Cells |
| spelling | doaj-art-afdec3b48b364548bebf34cf94e9c6a62025-08-20T04:00:54ZengMDPI AGCells2073-44092025-07-011415116610.3390/cells14151166Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE CellsRebecca D. Miller0Isaac Mondon1Charles Ellis2Anna-Marie Muir3Stephanie Turner4Eloise Keeling5Htoo A. Wai6David S. Chatelet7David A. Johnson8David A. Tumbarello9Andrew J. Lotery10Diana Baralle11J. Arjuna Ratnayaka12School of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Human Development and Health, Institute for Developmental Sciences Building, Tremona Road, Southampton, Hampshire SO16 6YD, UKBiomedical Imaging Unit, University of Southampton, MP12, Tremona Road, Southampton SO16 6YD, UKBiomedical Imaging Unit, University of Southampton, MP12, Tremona Road, Southampton SO16 6YD, UKSchool of Biological Sciences, Faculty of Environmental and Life Sciences, Life Sciences Building 85, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKSchool of Human Development and Health, Institute for Developmental Sciences Building, Tremona Road, Southampton, Hampshire SO16 6YD, UKSchool of Clinical and Experimental Sciences (CES), Faculty of Medicine, University of Southampton, MP 806, Tremona Road, Southampton SO16 6YD, UKRNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells.https://www.mdpi.com/2073-4409/14/15/1166retinal pigment epithelium (RPE)RNA-seqphotoreceptor outer segments (POS)traffickingproteostasisage-related macular degeneration (AMD) |
| spellingShingle | Rebecca D. Miller Isaac Mondon Charles Ellis Anna-Marie Muir Stephanie Turner Eloise Keeling Htoo A. Wai David S. Chatelet David A. Johnson David A. Tumbarello Andrew J. Lotery Diana Baralle J. Arjuna Ratnayaka Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells Cells retinal pigment epithelium (RPE) RNA-seq photoreceptor outer segments (POS) trafficking proteostasis age-related macular degeneration (AMD) |
| title | Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells |
| title_full | Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells |
| title_fullStr | Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells |
| title_full_unstemmed | Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells |
| title_short | Whole RNA-Seq Analysis Reveals Longitudinal Proteostasis Network Responses to Photoreceptor Outer Segment Trafficking and Degradation in RPE Cells |
| title_sort | whole rna seq analysis reveals longitudinal proteostasis network responses to photoreceptor outer segment trafficking and degradation in rpe cells |
| topic | retinal pigment epithelium (RPE) RNA-seq photoreceptor outer segments (POS) trafficking proteostasis age-related macular degeneration (AMD) |
| url | https://www.mdpi.com/2073-4409/14/15/1166 |
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