Simple repetitive RNAs are required for fertility and viability in Drosophila melanogaster

For proper cell function, DNA must be finely packaged into ‘chromatin’, which is the complex packaging of DNA with proteins and RNA. One state of gene-poor chromatin, termed heterochromatin, is a dense state of chromatin enriched for repeated RNA sequences and defined by repressive histone marks, and is critical for telomere maintenance, pericentromeric cohesion, meiotic segregation, and double strand break repair. Whether or not functions of heterochromatin stem from the underlying DNA, proteins or RNA, or a combination of all three, has largely been unexplored. Recent advances in sequencing technology have identified thousands of RNAs that do not code for proteins, termed non-coding RNAs (ncRNAs), and intriguingly some of them function to modify chromatin organization. Due to difficulties in sequencing RNAs that originate from heterochromatin, most ncRNAs described thus far were found to come from non-heterochromatic regions. Furthermore, due to limitations in studying functionality of ncRNAs, most studies were performed in-vitro and not in whole animals. Therein there lies a great need to identify which heterochromatic RNAs are transcribed and determine if any of these exhibit functions in whole animals.Heterochromatin is composed primarily of repeated DNA sequences, such as transposable elements (TEs), ribosomal DNA and satellites, which are blocks of tandem repeats varying in size from a few repeats to several megabases. Here I identify repetitive satellite ncRNAs that are transcribed from heterochromatin in Drosophila melanogaster. The most abundant forms of these satellite ncRNAs contain AAGAG(n) tandem repeats, are maternally loaded and associate with the earliest forms of heterochromatin, which suggests that they have roles in heterochromatin formation. These satellite ncRNAs are also found in later stages of Drosophila development in virtually every cell of embryos and larvae, and in addition are enriched in neural tissue and germline cells. In male testes they are enriched in the spermatocytes cells, which prompted us to determine if AAGAG(n) RNA containing satellites contribute to male fertility. We demonstrate that decreasing the levels of these satellites in spermatocytes causes 100% sterility by preventing the production of mature sperm, likely by affecting chromatin organization. These ncRNAs also bind an abundance of proteins involved in or present in spermatogenesis, suggesting that AAGAG(n) containing satellite RNAs affect organization and functions of these proteins. We also find that AAGAG(n) RNA containing satellites bind heterochromatic proteins, and furthermore provide preliminary evidence that these satellites are necessary for viability. This study thus provides not only some of the first evidence that satellite RNAs are transcribed in Drosophila, but that they can also exhibit critical functions. Contrary to the notion that heterochromatin serves mainly to silence RNA, this study suggests that RNAs derived from heterochromatin are critical interactors in the cell. We also identified long non-coding RNAs (lncRNAs) that associate with heterochromatin proteins and provide detailed subcellular and sub-nuclear distribution of these lncRNAs throughout development. Some of these lncRNAs are expressed only from certain tissues, suggesting they have roles in development. This is an important database that will provide researchers seeking to study these individual lncRNAs a foundation from which to begin to elucidate functions