{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/132740"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/132740","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Understanding small RNA-mediated post-transcriptional regulation of Salmonella pathogenicity island 1 activator HilA in Salmonella typhimurium","abstract":"Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. I compared two high-throughput approaches for sRNA target identification. I developed a modified MS2-Affinity Purification and Sequencing (MAPS) protocol and employed rGRIL-Seq (reverse Global sRNA Target Identification by Ligation and Sequencing). These methods used hilA 5' UTR tagging to capture cognate sRNA regulators. Comparative analysis revealed distinct advantages and limitation of each approach for dissecting target-focused sRNA discovery. rGRIL-Seq analysis revealed the potential sponge function of hilA 5’ UTR, identifing an sRNA-mediated SPI-1 post-transcriptional regulatory network composed of four sRNAs: Stnc2090, Stnc290, Stnc540 and Stnc3630, the results also defined sRNA InvR binding site within the hilA ribosome binding site region. Further genetic and molecular characterization showed that InvR directly base-pairs with hilA mRNA to prevent translation initiation, functioning to counteract HilA overactivation by HilD. This work revealed the structure of sRNA regulatory networks controlling SPI-1. Expression of master regulators such as HilD is extensively targeted by multiple sRNAs, while downstream effectors like HilA receive minimal direct post-transcriptional regulation. This pattern likely reflects evolutionary optimization for regulatory efficiency. The limited direct hilA regulators may result from evolutionary pressure against inappropriate activation of this essential virulence factor. These results advance our understanding of post-transcriptional regulatory network in SPI-1 virulence gene expression and demonstrate the sophisticated molecular strategies employed by Salmonella to integrate environmental signals for precise virulence gene-expression control.","abstract_html":"Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. I compared two high-throughput approaches for sRNA target identification. I developed a modified MS2-Affinity Purification and Sequencing (MAPS) protocol and employed rGRIL-Seq (reverse Global sRNA Target Identification by Ligation and Sequencing). These methods used hilA 5' UTR tagging to capture cognate sRNA regulators. Comparative analysis revealed distinct advantages and limitation of each approach for dissecting target-focused sRNA discovery. rGRIL-Seq analysis revealed the potential sponge function of hilA 5’ UTR, identifing an sRNA-mediated SPI-1 post-transcriptional regulatory network composed of four sRNAs: Stnc2090, Stnc290, Stnc540 and Stnc3630, the results also defined sRNA InvR binding site within the hilA ribosome binding site region. Further genetic and molecular characterization showed that InvR directly base-pairs with hilA mRNA to prevent translation initiation, functioning to counteract HilA overactivation by HilD. This work revealed the structure of sRNA regulatory networks controlling SPI-1. Expression of master regulators such as HilD is extensively targeted by multiple sRNAs, while downstream effectors like HilA receive minimal direct post-transcriptional regulation. This pattern likely reflects evolutionary optimization for regulatory efficiency. The limited direct hilA regulators may result from evolutionary pressure against inappropriate activation of this essential virulence factor. These results advance our understanding of post-transcriptional regulatory network in SPI-1 virulence gene expression and demonstrate the sophisticated molecular strategies employed by Salmonella to integrate environmental signals for precise virulence gene-expression control.","abstract_has_math":false,"creators":["Hou, Yutong"],"institution":"University of Illinois Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Microbiology","degree_department":null,"school":null,"contributors":["Vanderpool, Carin K.","Slauch, James M.","Whitaker, Rachel","Mera, Paola"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-12","date_published":"2025-12","updated_at":"2026-07-22T22:25:07Z","subjects":["Salmonella Pathogenicity Island","Salmonella","sRNA","Post-transcriptional regulation","HilA","Gene regulation","Virulence"],"languages":["en"],"rights":["Copyright 2025 Yutong Hou"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/132740","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Vanderpool, Carin K.","Slauch, James M.","Whitaker, Rachel","Mera, Paola"]},{"key":"dc:creator","label":"Author","values":["Hou, Yutong"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2025-12","2025-09-22"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Salmonella Pathogenicity Island","Salmonella","sRNA","Post-transcriptional regulation","HilA","Gene regulation","Virulence"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2025 Yutong Hou"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/132740"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. I compared two high-throughput approaches for sRNA target identification. I developed a modified MS2-Affinity Purification and Sequencing (MAPS) protocol and employed rGRIL-Seq (reverse Global sRNA Target Identification by Ligation and Sequencing). These methods used hilA 5' UTR tagging to capture cognate sRNA regulators. Comparative analysis revealed distinct advantages and limitation of each approach for dissecting target-focused sRNA discovery. rGRIL-Seq analysis revealed the potential sponge function of hilA 5’ UTR, identifing an sRNA-mediated SPI-1 post-transcriptional regulatory network composed of four sRNAs: Stnc2090, Stnc290, Stnc540 and Stnc3630, the results also defined sRNA InvR binding site within the hilA ribosome binding site region. Further genetic and molecular characterization showed that InvR directly base-pairs with hilA mRNA to prevent translation initiation, functioning to counteract HilA overactivation by HilD. This work revealed the structure of sRNA regulatory networks controlling SPI-1. Expression of master regulators such as HilD is extensively targeted by multiple sRNAs, while downstream effectors like HilA receive minimal direct post-transcriptional regulation. This pattern likely reflects evolutionary optimization for regulatory efficiency. The limited direct hilA regulators may result from evolutionary pressure against inappropriate activation of this essential virulence factor. These results advance our understanding of post-transcriptional regulatory network in SPI-1 virulence gene expression and demonstrate the sophisticated molecular strategies employed by Salmonella to integrate environmental signals for precise virulence gene-expression control.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-12-01","The student, Yutong Hou, accepted the attached license on 2025-09-19 at 11:51.","The student, Yutong Hou, submitted this Dissertation for approval on 2025-09-19 at 11:55.","This Dissertation was approved for publication on 2025-09-22 at 16:12.","DSpace SAF Submission Ingestion Package generated from Vireo submission #22806 on 2026-02-19 at 20:08:18"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Understanding small RNA-mediated post-transcriptional regulation of Salmonella pathogenicity island 1 activator HilA in Salmonella typhimurium"]}]}],"canonical_facts":{"dc:contributor":["Vanderpool, Carin K.","Slauch, James M.","Whitaker, Rachel","Mera, Paola"],"dc:creator":["Hou, Yutong"],"dc:date":["2025-12","2025-09-22"],"dc:description":["Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. I compared two high-throughput approaches for sRNA target identification. I developed a modified MS2-Affinity Purification and Sequencing (MAPS) protocol and employed rGRIL-Seq (reverse Global sRNA Target Identification by Ligation and Sequencing). These methods used hilA 5' UTR tagging to capture cognate sRNA regulators. Comparative analysis revealed distinct advantages and limitation of each approach for dissecting target-focused sRNA discovery. rGRIL-Seq analysis revealed the potential sponge function of hilA 5’ UTR, identifing an sRNA-mediated SPI-1 post-transcriptional regulatory network composed of four sRNAs: Stnc2090, Stnc290, Stnc540 and Stnc3630, the results also defined sRNA InvR binding site within the hilA ribosome binding site region. Further genetic and molecular characterization showed that InvR directly base-pairs with hilA mRNA to prevent translation initiation, functioning to counteract HilA overactivation by HilD. This work revealed the structure of sRNA regulatory networks controlling SPI-1. Expression of master regulators such as HilD is extensively targeted by multiple sRNAs, while downstream effectors like HilA receive minimal direct post-transcriptional regulation. This pattern likely reflects evolutionary optimization for regulatory efficiency. The limited direct hilA regulators may result from evolutionary pressure against inappropriate activation of this essential virulence factor. These results advance our understanding of post-transcriptional regulatory network in SPI-1 virulence gene expression and demonstrate the sophisticated molecular strategies employed by Salmonella to integrate environmental signals for precise virulence gene-expression control.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-12-01","The student, Yutong Hou, accepted the attached license on 2025-09-19 at 11:51.","The student, Yutong Hou, submitted this Dissertation for approval on 2025-09-19 at 11:55.","This Dissertation was approved for publication on 2025-09-22 at 16:12.","DSpace SAF Submission Ingestion Package generated from Vireo submission #22806 on 2026-02-19 at 20:08:18"],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/132740"],"dc:language":["en"],"dc:rights":["Copyright 2025 Yutong Hou"],"dc:subject":["Salmonella Pathogenicity Island","Salmonella","sRNA","Post-transcriptional regulation","HilA","Gene regulation","Virulence"],"dc:title":["Understanding small RNA-mediated post-transcriptional regulation of Salmonella pathogenicity island 1 activator HilA in Salmonella typhimurium"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Microbiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:07Z"}