Endocrine Abstracts

Objectives: The thyroid hormone receptor α (TRα) is crucial for organ development, regulation of growth, heart rate and energy homeostasis. In humans, THRA encodes for the two major TRα isoforms, TRα1 and TRα2. TRα2 is generated by alternative splicing and is unable to bind T3 and thus antagonizes TRα1 signaling. Therefore, the physiological effects of TRα require strictly controlled spatiotemporal expression of TRα isoforms. However, the mechanisms that regulate expression and cellular abundance of TRα isoforms are largely unknown. To fill this gap of knowledge we looked beyond classical concepts of post-transcriptional regulation and focused on RNA guanine-quadruplexes (RNA-G4s). RNA-G4s can act as cis-regulatory elements on translation and alternative splicing and RNA-G4s are present in THRA. Thus, we hypothesize that RNA-G4s play a role in the regulation of translation and alternative splicing of TRα isoforms.

Methods and Results: We used in silico predictions tools (QGRS-Mapper) and analysis of genome wide G4-sequencing data to identify G4-forming sequences in the 5’-UTR of TRα 58 bp upstream to the translational start site and two G4-forming sequences in intron 9, 148 bp downstream to the alternative splice site in exon 9. All three RNA-G4s are highly conserved among mammals. Circular dichroism spectroscopy and FRET-melting analysis confirmed formation of stable parallel RNA-G4s, as well as structural destabilization by specific point mutations. To determine the effect of the RNA-G4 in the 5’-UTR on translation we established a dual-luciferase reporter assay. Destabilization of the RNA-G4 by point mutations increased translation efficiency more than 3-fold. Remarkably, qRT-PCR confirmed that transcription was unaffected, demonstrating that the wild-type RNA-G4 sequence indeed represses translation of TRα1 and TRα2. We used the RG6-splicing reporter system to show that the two G4 sequences in intron 9 affect alternative splicing by increasing exon inclusion up to 25±3% compared to 5±1% using a vector with G4-disrupting mutations. Co-transfection with the splicing factor ASF/SF2 further increased exon inclusion up to 39±3%, but only in cells with functional RNA-G4s, demonstrating that the G4s serve as cis-regulatory factors controlling alternative splicing of TRα.

Conclusions and Outlook: These data provide novel mechanistical evidence that RNA-G4s regulate translation and alternative splicing of TRα isoforms. Currently, we test G4 ligands to modify TRα expression pharmacologically and unravel the physiological and cell-specific relevance of these G4s by using a CRISPR/Cas9 approach. Identification of tissue-specific G4-interacting factors is the next step as this might explain the spatiotemporal differences in TRα isoform expression.