No abstract

Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.

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 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 ␣-helix/␤-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 binding of proteins to RNA is a fundamental aspect of biology that interferes with most aspects of gene expression and cellular functions. The presence of various binding motifs defines the group of RNA binding proteins (1). Commonly found RNA binding domains include the RNA recognition motif (RRM), 3 the double-stranded RNA binding domain, the Piwi Argonaut and Zwille domain, and the heterogeneous nuclear ribonucleoprotein K homology domain. The most prominent RNA binding domain is the RRM that is found in ϳ2% of human proteins (2). The RRM is composed of two consensus sequences RNP2 and RNP1 that contain aromatic residues important for RNA binding. In other RNA binding motifs, such as the PUA (pseudouridine synthase and archaeosine transglycosylase) and OB-fold (oligonucleotide/oligosaccaride binding fold), the RNA interacts with the ␤-sheets that form pseudobarrels (3). The general composition of the PUA domain is reminiscent of the OB-fold, a nucleic acid binding motif that displays only a low degree of sequence similarity between its members. The OB-fold consists of two three-stranded antiparallel ␤-sheets, where strand 1 is shared by both sheets. The individual ␤-sheets can be separated by protein parts of different length, which makes the identification based on primary structure difficult (4). The ␤-sheets in the PUA and OB-folds form a ligand binding surface that can bind to nucleic acids through aromatic stacking, hydrogen bonding, as well as polar and hydrophobic interactions. The so-far unexplained RNA binding activities of proteins such as apontic (5) demonstrate that not all RNA binding domains have been described.One of the potentially new RNA binding domains is the YTH (YT521 homology) domain. The YTH domain is highly conserved during evolution and was identified by comparing all known protein sequences with the splicing factor YT521-B (6). The domain is found only in eukaryotes and is abundant in plants. The YTH domain can be between 100 and 150 amino acids in size and is characterized by 14 invariant and 19 highly conserved residues. It is predicted to contain ...