There is a fundamental gap in understanding the function of small RNAs that constitute a large part of human gene expression. For example, we do not know how the loss of small nucleolar RNA (snoRNA) expression contributes to the Prader-Willi syndrome, the most frequent genetic cause for life-threatening obesity. Our long-term goal is to elucidate the regulation of alternative splicing by small RNAs that are estimated to comprise more than 40% of human gene expression. The objective of this application is to determine which alternative splicing patterns are influenced by the snoRNAs missing in individuals with Prader-Willi syndrome and to understand the molecular basis by which one of these snoRNAs, HBII-52, influences alternative splicing patterns. The central hypothesis is that snoRNAs missing in individuals with Prader- Willi syndrome regulate alternative splicing by masking splicing regulatory elements on pre- mRNAs. Thus these snoRNAs regulate expression of numerous genes by changing their alternative splicing patterns. The rationale for the proposed research is that the genes regulated by these snoRNAs represent drug targets for treatment of the Prader-Willi syndrome. This proposal is therefore relevant to the NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human disease. Guided by our first published report that the snoRNA HBII-52 influences alternative splicing and additional strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify the pre-mRNAs that are regulated by the snoRNAs missing in people with Prader-Willi syndrome;and 2) Determine how HBII-52 regulates alternative splice site usage and test alternatives for its expression. Under the first aim, changes in pre-mRNA processing will be determined after ectopically expressing individual snoRNAs using bioinformatic predictions and splice-site sensitive DNA arrays. Under the second aim, we will identify the repressor activity that competes with the snoRNA HBII-52 and determine its mode of action, using an established in vitro system. The proposed work is innovative, because it shows a complete new role for snoRNAs in the regulation of alternative pre-mRNA splicing. It may well change the current `textbook knowledge'that snoRNAs only regulate non-mRNAs. The proposed research is significant because it would show a novel role of snoRNAs, identify the molecular defects in Prader-Willi syndrome and help to predict the influence of small RNAs on splice site selection.

Public Health Relevance

The proposed studies delve into an under-investigated area of gene expression and have the potential applicability to understand the molecular cause of the Prader-Willi Syndrome and potentially other forms of inherited obesity. The proposed research is relevant for public health, because it investigates a new mechanism of human gene expression. Understanding this mechanism is the basis to improve diagnostics and therapeutic options for human diseases caused by defects in pre-mRNA processing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Bender, Michael T
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University of Kentucky
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Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta et al. (2016) Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing. Proc Natl Acad Sci U S A 113:E1625-34
Falaleeva, Marina; Surface, Justin; Shen, Manli et al. (2015) SNORD116 and SNORD115 change expression of multiple genes and modify each other's activity. Gene 572:266-73
Shen, Manli; Falaleeva, Marina; Korotkova, Natalia et al. (2015) Direct cloning of double-stranded RNAs. Methods Mol Biol 1296:53-64
Convertini, Paolo; Shen, Manli; Potter, Philip M et al. (2014) Sudemycin E influences alternative splicing and changes chromatin modifications. Nucleic Acids Res 42:4947-61
Falaleeva, Marina; Stamm, Stefan (2013) Processing of snoRNAs as a new source of regulatory non-coding RNAs: snoRNA fragments form a new class of functional RNAs. Bioessays 35:46-54
Zhang, Zhaiyi; Convertini, Paolo; Shen, Manli et al. (2013) Valproic acid causes proteasomal degradation of DICER and influences miRNA expression. PLoS One 8:e82895
Zhang, Zhaiyi; Falaleeva, Marina; Agranat-Tamir, Lily et al. (2013) The 5' untranslated region of the serotonin receptor 2C pre-mRNA generates miRNAs and is expressed in non-neuronal cells. Exp Brain Res 230:387-94
Convertini, Paolo; Zhang, Jiayu; de la Grange, Pierre et al. (2013) Genome wide array analysis indicates that an amyotrophic lateral sclerosis mutation of FUS causes an early increase of CAMK2N2 in vitro. Biochim Biophys Acta 1832:1129-35
Kelemen, Olga; Convertini, Paolo; Zhang, Zhaiyi et al. (2013) Function of alternative splicing. Gene 514:1-30
Shen, Manli; Bellaousov, Stanislav; Hiller, Michael et al. (2013) Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure. Nucleic Acids Res 41:3819-32

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