Obesity is a major and growing health problem. Mouse studies and the development of weight-loss drugs like fenfluramine–phentermine (fen–phen) validated the serotonin receptor 2C (HTR2c) protein as an anti-obesity drug target (1
). HTR2c is a G-protein–coupled receptor located on the X chromosome. As shown in , exon Vb is alternatively spliced. Only when this exon is included, a functional receptor can be made, as skipping of the exon generates a frame shift, which likely generates a mRNA, which is not translated into protein and is likely degraded.
Figure 1. Screen to identify substances that promote exon Vb inclusion. (A) Schematic overview of the HTR2c gene. The dot indicates the start codon. The shaded area is used to construct the fluorescence-based screening reporter genes. The dotted area containing (more ...)
The receptor pre-mRNA undergoes A → I editing in at least five editing sites located in exon Vb, together with alternative splicing generating at least 25 isoforms.
The inclusion of exon Vb can be achieved in two known ways: (i) changing one or more of five adenosines in exon Vb to inosine using the deaminases ADAR (adenosine deaminase acting on RNA) (2
); or (ii) by promoting exon Vb inclusion via processed small nucleolar RNAs (psnoRNAs) (3
) without any change in mRNA sequence. The inosines generated by ADAR are interpreted as guanosines by the ribosome. Therefore, the edited versions of the HTR2c have a different protein sequence. This difference in composition has a functional effect: the edited versions of the receptor are less active than the non-edited versions, as the coupling efficiency towards the G-protein is reduced (5
). Mice lacking HTR2c expression are hyperphagic and obese (6
). Although re-introducing the non-edited receptor reverts this phenotype, introduction of the fully edited HTR2c does not revert the hyperphagic behaviour (8
), suggesting that expression of the non-edited most active receptor is important to prevent hyperphagia.
The Prader–Willi syndrome is the most frequent genetic cause for obesity in humans. Genetic data indicate that the loss of regulatory RNAs generated from the HBII-52 and -85 clusters cause the disease (10
). The HBII-52 and -85 loci encode RNAs that structurally resemble snoRNAs, but the protein complexes they form are biochemically distinct from canonical small nucleolar ribonucleoprotein (snoRNPs) (3
). Furthermore, the HBII-52 and -85 loci express shorter RNAs, termed psnoRNAs (13
). HBII-52–derived psnoRNAs change alternative splicing of the serotonin receptor 2C. This promotes the formation of an mRNA encoding a protein isoform that has the strongest response to serotonin (3
). Collectively, these data show that the HTR2c pre-mRNA processing controls appetite in vertebrates.
The use of RNA-binding antibiotics, such as gentamycin, chloramphenicol and tetracycline, demonstrates that drugs can target RNA molecules (15–17
). The target for these antibiotics is bacterial ribosomal RNA that is highly structured. Most pre-mRNAs have short rapidly changing secondary structures, if they have structures at all. In contrast, the HTR2c pre-mRNA is edited at several positions (C). This is only possible when the pre-mRNA forms a transiently stable secondary structure in vivo
, which is necessary for its editing by ADAR enzymes that need double-stranded RNA as substrates (18
). This scenario is unique, as pre-mRNA processing likely occurs concomitant with transcription, and the structures of pre-mRNAs in vivo
are generally not known (19
). As the pre-mRNA is structured and at least transitionally stable in vivo
, it presents defined molecular binding sites, making the HTR2c pre-mRNA a potential drug target similar to the structured ribosomal RNAs recognized by antibiotics.
Figure 6. Structure probing of the exon Vb region. (A) Schematic representation of the probe used. 5B is the regulated exon and DS and PS are proximal and distal splice sites, respectively. The line indicates the SP6 polymerase-generated probe, and the arrow indicates (more ...)
Most substances that change alternative splicing work indirectly. They influence mainly post-translational modifications, such as phosphorylation (21
), or cause a change in regulatory factors (22
) [reviewed in (23
)]. However, pre-mRNA riboswitches have been identified in bacteria, and a vitamin B1 riboswitch regulating alternative splicing has been found in fungi (24
). This demonstrates that pre-mRNA splicing can be directly regulated by interaction of pre-mRNA with small molecules.
Given the importance of proper alternative splicing regulation for human health, screening systems for substances that change alternative splicing have been developed (25
Here, we report the identification of pyrvinium pamoate as a drug that promotes inclusion of the alternative exon of the serotonin receptor 2C through direct binding to the pre-mRNA. Pre-mRNA binding causes a conformational change, which makes the regulated splice site more accessible to the splicing machinery, promoting exon inclusion.