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Alternative RNA splicing of Rps6ka3 downstream of PDGFR signaling in the facial mesenchyme

Award Number: F31DE035358
ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 02/01/2027
PERIOD OF PERFORMANCE END DATE: 01/31/2028

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Alternative RNA splicing of Rps6ka3 downstream of PDGFR signaling in the facial mesenchyme - Project Summary Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth differences. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays critical roles in this process in humans and mice. Variants in human PDGFRA are associated with non- syndromic cleft lip/palate, whereas those in PDGFRB cause syndromes characterized by facial dysmorphism. Mice with conditional ablation of Pdgfra and/or Pdgfrb in the neural crest cell (NCC) lineage phenocopy these human syndromes and present with midline facial defects. Previous sequencing of maxillary process (MxP) mesenchyme RNA from E11.5 mouse embryos lacking Pdgfra and/or Pdgfrb in the NCC lineage demonstrated that alternative RNA splicing (AS) is the predominant mechanism of gene expression regulation downstream of PDGFR signaling in this setting. Among AS events, there was an enrichment for transcripts encoding protein serine/threonine kinases within the phosphatidylinositol 3-kinase (PI3K) signaling pathway, including Rps6ka3 (encoding Rsk2). Mutations in human RPS6KA3 cause Coffin-Lowry Syndrome, which is characterized by distinctive facial features such as a high, narrow palate. Similarly, Rps6ka3-null mice display hypoplasia of several craniofacial bones. Despite the critical role of Rps6ka3 in craniofacial development, regulation of Rps6ka3 gene expression and the effects of different isoforms on cell activity have not been explored. The goal of this proposal is to determine the molecular mechanisms by which PDGFRa-induced AS of Rps6ka3 affects translation to ultimately regulate downstream intracellular signaling and facial mesenchyme proliferation. First, the effect of AS of Rps6ka3 on translation will be assessed. The order of included exons in Rps6ka3 will be determined using targeted long-read sequencing of E11.5 MxP mesenchyme from wild-type embryos or those lacking Pdgfra in the NCC lineage. The expression levels of Rps6ka3 transcript and protein isoforms will be assessed via qRT-PCR and western blotting (WB), respectively, in mouse embryonic palatal mesenchyme (MEPM) cells derived from embryos with these same genotypes. The effect of AS of Rps6ka3 on translation will be evaluated using polysome profiling and, separately, luciferase reporter assays in MEPM cells. Second, the effect of AS of Rps6ka3 on substrate phosphorylation and proliferation in palatal mesenchyme cells will be determined. To establish the consequences of AS of Rps6ka3 on Rsk2 kinase activity, phosphorylation levels of craniofacial-relevant substrates Atf4, Fgfr1 and Gsk3b will be assessed via WB. Separately, plasmids encoding different Rps6ka3 isoforms will be overexpressed, and Rsk2 substrate phosphorylation and cellular proliferation will be assessed via WB and Ki67 immunofluorescence analysis, respectively. These studies will identify the mechanism and consequences of growth factor-mediated AS on downstream intracellular signaling and cell activity in the palatal mesenchyme, thus providing significant insight into gene expression regulation during mammalian craniofacial development.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $43,880 )
2026	2026	THE REGENTS OF THE UNIVERSITY OF COLORADO	13001 E 17TH PL STE F428	AURORA	CO	80045	ADAMS	USA	Oral Diseases and Disorders Research	000	1	8/12/2026	NEW	$43,880
														Subtotal = $43,880

Grand Total All Awards = $43,880

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Alternative RNA splicing of Rps6ka3 downstream of PDGFR signaling in the facial mesenchyme

Award Number: F31DE035358

ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 02/01/2027

PERIOD OF PERFORMANCE END DATE: 01/31/2028

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