{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:58:02Z","timestamp":1782280682243,"version":"3.54.5"},"posted":{"date-parts":[[2025,11,18]]},"group-title":"Bioinformatics","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T00:00:00Z","timestamp":1763424000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"name":"Intramural Research Program of the NIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases.","award":["ZIA-AR041205 to M.H."],"award-info":[{"award-number":["ZIA-AR041205 to M.H."]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  PAR-CLIP is a widely used method for identifying binding sites of RNA-binding proteins (RBPs) transcriptome-wide. A characteristic T-to-C transition in the sequenced cDNA pinpoints the site of RBP-RNA crosslinking and is induced by the use of a photoreactive uridine analogue, 4-thiouridine (4SU). As with other systems-wide methods, PAR-CLIP, too, is prone to false discoveries as the T-to-C signal might result from systematic noise, pre- existing SNPs, and PCR errors. Therefore, rigorous statistical methods are required for analyzing PAR-CLIP data. The few existing tools to analyze PAR-CLIP data lack updates and sufficient documentation, and often fail to process current higher-depth sequencing data. Here we report PCLIPtools, a lightweight, customizable suite for analyzing PAR-CLIP data. PCLIPtools considers the read depth, T-to-C transitions, and the other mutations to statistically estimate high-confidence interaction sites. Benchmarking shows that PCLIPtools identifies more functionally significant targets than the current standard tool, PARalyzer, without losing high-confidence sites and outperforming it in runtime. Exploratory analyses show PCLIPtools\u2019 specific targets are enriched for read depth and T-to-C conversion, supporting their validity. With simplicity, robustness, and speed, PCLIPtools improves the precision of PAR-CLIP data analysis while being accessible to experimental RNA biologists. PCLIPtools can be found on github (\n                  <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/paulahsan\/pcliptools\">https:\/\/github.com\/paulahsan\/pcliptools<\/jats:ext-link>\n                  ).\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"688892v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.11.17.688892","type":"posted-content","created":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T11:10:15Z","timestamp":1763464215000},"source":"Crossref","is-referenced-by-count":1,"title":["PCLIPtools: A Robust Framework for Identifying RNA-Protein Interaction Sites from PAR-CLIP experiments"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6591-3164","authenticated-orcid":false,"given":"Ahsan H.","family":"Polash","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4336-6518","authenticated-orcid":false,"given":"Markus","family":"Hafner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025112001350626000_2025.11.17.688892v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-080111-092106"},{"key":"2025112001350626000_2025.11.17.688892v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3813"},{"key":"2025112001350626000_2025.11.17.688892v1.3","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab011"},{"key":"2025112001350626000_2025.11.17.688892v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2020.03.011"},{"key":"2025112001350626000_2025.11.17.688892v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2010.12.011"},{"key":"2025112001350626000_2025.11.17.688892v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymeth.2005.07.018"},{"key":"2025112001350626000_2025.11.17.688892v1.7","doi-asserted-by":"publisher","DOI":"10.1126\/science.1090095"},{"key":"2025112001350626000_2025.11.17.688892v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3810"},{"key":"2025112001350626000_2025.11.17.688892v1.9","doi-asserted-by":"crossref","unstructured":"Konig, J. , Zarnack, K. , Rot, G. , Curk, T. , Kayikci, M. , Zupan, B. , Turner, D.J. , Luscombe, N.M. and Ule, J . 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(2024) <em>MATRIN3<\/em> deficiency triggers autoinflammation via cGAS-STING activation. bioRxiv, 2024.2004.2001.587645.","DOI":"10.1101\/2024.04.01.587645"},{"key":"2025112001350626000_2025.11.17.688892v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-10432-5"},{"key":"2025112001350626000_2025.11.17.688892v1.27","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/s12860-023-00488-z","article-title":"Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer","volume":"24","year":"2023","journal-title":"BMC Mol Cell Biol"},{"key":"2025112001350626000_2025.11.17.688892v1.28","doi-asserted-by":"crossref","unstructured":"Martin, M. 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