The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an α-helix/β-sheet structure. Here we show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single-stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA binding ability to a new class of proteins that are found in all eukaryotic organisms.

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader–Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a “splicing” or “messenger ribonucleoprotein code” that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites.