PROJECT SUMMARY The intent of this proposed research is to contribute to the basic understanding of the structure and function of RNA that is expressed from tandemly repeated regions of the human genome. Due to recent advances in sequencing of a complete human genome, the tandemly repeated DNA sequences residing within the centromere and the adjacent regions (the pericentromere) have only recently been fully characterized. It is known that these abundant tandem repeat sequences, which constitute 6.2% of the human genome, typically remain silent or are only expressed at low levels in normal cells. However, in cancer cells, HSATII, a tandemly repeated pericentric satellite sequence, is aberrantly transcribed into RNA. In these cells, HSATII RNA accumulates in the nucleus, adjacent to its site of transcription, where it recruits, and potentially sequesters, nuclear regulatory proteins. While HSATII DNA is found on many different human chromosomes, only a few of these locations transcribe HSATII RNA, suggesting that the regulation and sequence composition may vary from one chromosome to another. With the new resource of a complete sequence map of these pericentric regions, we can now characterize the chromosomal locations from which HSATII RNA is transcribed in cancer cells for the first time. In order to study expressed HSATII sequences, long-read sequencing of nuclear expressed RNA will be mapped to chromosome-specific HSATII variants. It is hypothesized that expressed HSATII loci will harbor unique sequence motifs. The hypothesis will be tested by mapping and characterizing expressed chromosome-specific HSATII variants (Aim 1) and by functional analysis of individual expressed sequence variants in normal cells (Aim 2). The phenotypic effect of HSATII nuclear accumulation will be accomplished by characterizing the structure of HSATII RNA and its capacity to bind nuclear regulatory proteins (Aim 3). All of the proposed research will be conducted by undergraduate students under the close supervision of the PI, in collaboration with other leaders in genomic and RNA structural analyses. Thus, the proposed project promises to engage and train undergraduate researchers in innovative genomics, cytological, and RNA structural probing techniques, which will propel future careers in genomics and biomedical research.