Identification of the First Telomerase RNA from the Filamentous Fungus Aspergillus oryzae and Related Organisms

Chromosomal ends are protected by telomeres, which are synthesized by the ribonucleoprotein telomerase. Telomerase consists of a reverse transcriptase protein (TERT) and an RNA (TR). The TR contains the template that helps elongate the telomere. While the TERT is highly conserved among organisms, the TR sequence is highly divergent, and thus difficult to identify. Through my experiments I found the previously unidentified TR gene sequence of the filamentous fungus Aspergillus oryzae. I experimentally determined the ends of this sequence, which then allowed for the sequence to be folded and have its secondary structure examined. When folded, the sequence contained the template within a single stranded region, a hallmark of all TRs. I searched the genomes of related organisms for similar sequences, ultimately finding 12 new TRs. These are the first telomerase RNAs identified in any filamentous fungi, and will provide insights into the structure and evolution of a complex RNA. Introduction The stability of genetic material is of the utmost importance to all cells. If the genetic material becomes compromised in any way, cellular function may be negatively affected, possibly even leading to cell death or the production of a cancerous cell (Loeb and Loeb, 2000). For this reason cells have evolved numerous intricate systems and structures that maintain the integrity of the genetic material (Hoeijmakers, 2001). One such adaptation that has evolved in eukaryotic cells to protect the end of a chromosome is the telomere. The term telomere, first coined by Hermann J. Müller, originates from the two Greek words, telo, meaning end, and mere, meaning part (Müller, 1938). In the 1930’s Müller (1938) found that the end of chromosomes did not possess deletions or inversions after irradiation due to the protective cap of the telomere (Chuaire, 2006). McClintock (1941) found that when chromosomes had their natural ends removed, the ends were more likely to fuse together, whereas those that maintained their natural ends would not. Some 40 years later Blackburn and Gall (1978) identified that the ends of the chromosomes from Tetrahymena thermophila consisted of 20-70 tandem repeats of the sequence 5’-TTGGGG-3’. These discoveries were the first of what would soon open up into the vast field of telomere biology, which would encompass everything from diseases to enzymes to the potential of reversing the aging process. One of the most prevalent telomeric repeats, present in all vertebrates, consists of the repeating nucleotide sequence 5’-TTAGGG-3’, while telomeres from other organisms are based on slightly different sequences (Meyne et al., 1989) (Table 1). For example, Caenorhabditis elegans has the telomeric repeat of 5’-TTAGGC-3’, while Saccharomyces cerevisiae uses 5’-TG2-3(TG)1-6-3’ (Wicky et al., 1996; Duffy and Chambers, 1996). The length of the ________________________________________________ *This author wrote the paper as a senior thesis for biology and received distinction under the direction of Dr. Karen Kirk. For example, Caenorhabditis elegans has the telomeric repeat of 5’-TTAGGC-3’, while Saccharomyces cerevisiae uses 5’-TG2-3(TG)1-6-3’ (Wicky et al., 1996; Duffy and Chambers, 1996). The length of the telomere also varies depending on the organism. In humans the telomere consists of about 250-1000 repeats, meaning that the length of the telomere is about 1500-6000 bp, while the telomere of T. thermophila consists of about 50 repeats, with the length of the telomere about 300-400 bp (Moyzis et al., 1988; Blackburn and Gall, 1978). Though the length of telomeres between organisms can be quite different, of more importance is the individual organism’s ability to maintain the length of its own telomeres. This is important due to the discoveries by Watson (1972) and Olovnikov (1973), which culminated in the idea known as the end replication problem (Verdun and Karlseder, 2007). End Replication Problem In order for a cell to divide into two daughter cells, the chromosomes must duplicate to accommodate the two cells. This duplication occurs through the process of DNA replication. The enzyme primase synthesizes an RNA primer that is complimentary to the parent strand. The DNA polymerase recognizes the RNA primer initiating synthesis of a new strand (Kornberg, 1984). Once DNA replication begins the RNA primer degrades. This is problematic because if the RNA primer is located at the 5’ terminus of the chromosome, a gap of nucleotides will be left after the RNA primer degrades. This replication therefore would leave a chromosome with a slightly shortened 5’ end and a 3’ overhang (Fig. 1). Thus with each successive replication of a chromosome, the chromosome would become shorter and shorter. Figure 1. The end replication problem The two parent strands of DNA (in blue with grey telomeres) have been used as templates to synthesize new complimentary strands (black). The new strands of DNA have the RNA primers (red) present at the 5’ ends that are used to initiate DNA replication. The primer degrades, producing a 3’ overhang. Some scientists believed that telomeres acted as a buffer to prevent the loss of genes interior to the chromosome, whose loss could have detrimental effects on the cell (Olovnikov, 1973). This idea is supported by Hayflick and Moorhead’s (1961) previous finding that after about 50 divisions human somatic cells are no longer viable. Harley et al. (1990) further supported this idea by showing that with each round of DNA replication that a cell undergoes there is a decrease of about 50 bp at the telomere. These findings Eukaryon, Vol. 9, March 2013, Lake Forest College Senior Thesis show the importance of telomeres and how they might be implicated in the aging of cells once they get too short. Further research on telomeres would lead to their importance as a capping mechanism through the recruitment of proteins that aid in the stability of the ends of chromosomes (O’Sullivan and Karlseder, 2010). T