The role of guanine-quadruplexes in cell type-specific transcriptional and post-transcriptional regulation of thyroid hormone receptors TRalpha1 and TRalpha2

• Applicant: Dr. Georg Sebastian Hönes
• Subject Area: Endocrinology, Diabetology, Metabolism
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525051195

Thyroid hormone (TH) is crucial for normal development, growth and regulation of important physiological functions. These pleiotropic effects are exerted by binding of TH to its TH receptors (TR) alpha (TRa) and beta. The gen THRA encodes for two main receptor isoforms TRa1 and TRa2, whereas the latter is generated by alternative splicing and is unable to bind TH. This counteracts TRa1 function. Therefore, the TH response of a certain cell-type is ultimately defined by expression of a distinct TR isoform. Even after 35 years of TR research the factors that regulate expression of TRa isoforms are still unknown. We suggest guanine-quadruplexes (G4) to play a decisive role in that context. G4s are important regulatory elements of gene expression that can be formed by DNA as well as RNA. In promoter regions they regulate transcription, can inhibit protein biosynthesis when appearing in the 5’UTR of mRNAs and are involved in the regulation of alternative splicing. In our project we want to investigate the role of G4s in organ-specific expression of TRa1 and TRa2. We analyzed THRA and identified two G4s in the promoter, one RNA-G4 in the 5’UTR and five RNA-G4-forming sequences between exon 9 and exon 10 (a region important for alternative splicing of TRa1 and TRa2), resulting in three independent subprojects:1. Determine the role of DNA-G4s in the promoter of THRA.2. Ascertain the regulatory function of the RNA-G4 the 5’UTR in translation of TRa. 3. Characterize the physiological relevance of RNA-G4s in alternative splicing of TRa. We will use FRET (Förster resonance energy transfer) and CD spectroscopy to determine the G4 structure and identify the minimal number of point mutations needed to disrupt G4-folding. Cell-based reporter assays (dual-luciferase assay and bichromatic splicing-reporter assay) will be conducted to assess the functionality of the G4 candidates and compare them to the corresponding mutated sequence. We will generate (CRIPSR/Cas) a FLAG-tag reporter cell line that enables us to investigate the effect of the desired G4s in the context of physiological expression of TRa1 and TRa2. G4-forming sequences a present in all cell types. Thus, it is necessary to identify cell- and organ-specific G4-bidning partners to address the cell type-specific differences in TRa isoform expression. G4-binding proteins will be identified with a pull-down assay combined with mass spectrometry (timsTOF). The function of the binding partners will be assessed in over-expression experiments and via gene silencing (RNA interference) using the reporter cell line. Achieving these goals will reveal the role of G4s in the regulation of TRa isoform expression. Identifying the hitherto unknown factors that regulate TRa expression is of high interest to the field of TR research, as it will expand our knowledge of local control of TH/TRa action.

• DFG Programme: Research Grants