Shivendra Kishore¹, Amit Khanna¹, Piotr Balwierz², Mihaela Stefan³, Robert D Nicholls³, Mihaela Zavolan² and Stefan Stamm¹

University of Kentucky, College of Medicine, Lexington, KY¹, Biocenter Basel, 4056 Basel, Switzerlan²d

Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, PA³

Background: The imprinted, paternally expressed SNURF-SNRPN-snoRNA locus that is not expressed by people with Prader Willi syndrome (PWS) contains several snoRNA clusters, including 47 copies of SNORD115 (formally HBII-52) and 24 copies of SNORD116 (HBII-85). SnoRNAs are small nuclear RNAs previously implicated in modification of ribosomal and transfer RNAs. They can interact with other RNAs using their antisense box elements. We recently showed that HBII-52 regulates alternative splicing of the serotonin receptor 5HT-2C (1).

Methods: Using overexpression of HBII-52, bioinformatics prediction, and array analysis, we identified another 7 splicing events that are regulated by HBII-52 snoRNA. The dependency of these splicing events on HBII-52 was confirmed by reporter gene analysis, binding site mutagenesis and was correlated with a dependency on HBII-52 in mouse models and human brain tissues.

Results/Discussion: Four of the targeted splicing events are in pre-mRNAs encoding nuclear proteins (C1orf77, CPSF4, PBRM1, TAF1) and three are in pre-mRNAs coding for signaling molecules (CRHR1, DPM2, RALGPS1). The analysis of the snoRNA binding sites indicate that there can be up to five base-pair mismatches between the snoRNA and its target, which is reminiscent to the low RNA binding requirements of miRNAs. Furthermore, the snoRNA binding site of the mRNA is adjacent to the predicted binding sites of splicing regulators SRp40, SRp55 and SC35. In its various targets, the snoRNA can either promote alternative exon skipping or inclusion.

To gain further insight into the mechanism of snoRNA action, we purified the protein particles that assemble around it in vitro. Surprisingly, we found AKT3, a member of the protein kinase B (PKB) family associated with the snoRNP. PKB is an insulin-regulated kinase acting on SR proteins such as SRp40, SRp55 and SC35, which are predicted to bind next to HBII-52 on target pre-mRNAs. The data suggest that HBII-52 links insulin signaling via PKB-dependent splicing factor phosphorylation to gene expression. The emerging knowledge of the splicing regulation allowed us to test whether small molecules can change splice site selection similar to HBII-52. We found that C6 ceramide treatment mimicks the effect of HBII-52 on exon inclusion of the TAF1 gene. Several of the target genes may correlate with clinical findings. For example, HBII-52 promotes inclusion of two adjacent alternative exons in the corticotropin releasing hormone receptor CRHR1. The lack of these exons generates a non-functional receptor. These data may explain central adrenal insufficiency in people with PWS (2), since they are predicted to express less of the full length CRHR1 receptor necessary for the stress response.

Conclusion: Our data show that HBII-52 snoRNA regulates processing of multiple mRNAs. The lack of HBII-52 deregulates the splicing events and will likely contribute to the phenotype.

References: (1) Kishore, S. and Stamm, S. (2006) Science, 311, 230-232., (2) de Lind van Wijngaarden, R.F., et al. (2008). J Clin Endocrinol Metab., 1649-1654

Edited: 02/09/2012