While most autosomal genes are expressed biallelically, genomic imprinting causes some to be expressed only from either the paternal or maternal chromosome. The human Chr15q11-q13 region is imprinted, leading to expression of the SNURF-SNRPN gene and downstream noncoding region from the paternal chromosome. This region is implicated in an important human disease, Prader-Willi Syndrome (PWS), which is associated with hypotonia, hypogonadism, hyperphagia and mild mental retardation. The long neuron-specific paternal transcript contains at least 148 exons and spans 470 kb. All paternal transcripts downstream of the SNRPN gene are noncoding and have been considered primarily as precursors for small RNAs. Since the smallest paternal deletions that cause PWS primarily remove the SNORD116 cluster of 29 similar snoRNAs, the most current published model is that SNORD116 deficiency is the primary cause of disease. How the snoRNAs in this region function in cells is completely unknown. These snoRNAs are of the ?box C/D? type, and such molecules are known to use complementary base pairing to promote specific 2'-O methylation of cellular RNAs, usually rRNAs or noncoding small RNAs. The SNORD116 RNAs are ?orphans?, as no targets have been identified so far, and bioinformatic approaches to identify or predict possible targets have failed. We propose to develop a novel technology to map 2'-OMe sites genomewide, and will apply it first in WT and PWS cells to find potential targets of SNORD116s. In the long term this method will allow studies on possible regulation and dynamic changes in rRNA modifications, and can be applied to the study of a number of other orphan snoRNAs.
We propose to develop a novel technology to map genomewide sites at which RNA molecules are modified by 2'-O methylation. These modifications are usually directed by small nucleolar RNAs (snoRNAs). A number of snoRNAs have been implicated in neurological disorders, including a cluster of them associated with Prader- Willi Syndrome. We will apply our new sequencing method to the study of the cellular target RNAs affected in this disorder.
