In most eukaryotes, the ends of chromosomes are capped by a series of short G-rich DNA repeats bound by a set of specific proteins. These so-called telomeres distinguish chromosome ends from DNA double-strand breaks and must be replenished continuously in dividing cells. Telomerase is the primary means of telomere elongation, and the enzyme is up-regulated in more than 80 percent of cancers. Conversely, mutations in telomerase components are linked to degenerative syndromes, including dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis. Our research in this area is aimed at deciphering the molecular mechanisms that underlie telomerase biogenesis and regulation.
A few years ago, we became interested in a genus of lizards in which several species are solely comprised of females that reproduce by true parthenogenesis. Despite considerable interest in the lizards′ ecology and evolution, little was known about the molecular mechanisms that permit females to clone themselves in the absence of males. Gaining a molecular understanding of the mechanisms that permit a switch to asexuality in some vertebrates carries the promise of furthering our understanding of meiosis and explaining why sexual reproduction is so widespread. At the very least, this line of research will be critical in determining whether parthenogenesis is a dead end or an important element in vertebrate evolution.
Telomerase Biogenesis and Regulation
While studying telomere end capping, we noticed that certain mutations in the protein Pot1 (protection of telomeres) cause dramatic telomere lengthening, which is indicative of the protein's role in regulating telomerase. Although the core components of telomerase have been known for some time, our understanding of telomerase assembly, function, and regulation is still limited. As part of the initial characterization of fission yeast telomerase, we established a precursor-product relationship between longer poly-adenylated RNAs and a shorter form of the RNA found in the active telomerase complex. We designed a series of experiments to examine how the precursors are trimmed to generate the shorter form of the RNA. These studies led us to the highly unexpected realization that the spliceosome has a function in 3'-end processing of this noncoding RNA. Instead of removing an intronic sequence in a two-step process, the first transesterification reaction alone generates the mature 3' end of telomerase RNA (Figure 1).
Termination after the first step of splicing would be highly detrimental if it occurred during intron removal due to the release of a truncated and nonfunctional RNA. For this reason, the two steps in splicing are normally tightly coupled, and at least for some introns, mutations at the 3' splice site block the first step of splicing, which is consistent with the spliceosome not starting a job it cannot finish. We are using biochemical assays and molecular genetics to gain insights into the mechanism by which the 5' exon is released after the first cleavage has occurred.
We are also examining other steps in telomerase biogenesis, such as transcriptional regulation, RNA modifications, and assembly of the holoenzyme. Downstream from these events, the questions of how telomerase is recruited specifically to the shortest telomeres and how the activity of the enzyme is regulated via posttranslational modifications remain to be answered. Our research goals in this area are guided by the belief that a better understanding of the dynamic interactions that occur at telomeres will ultimately enable us to identify compounds that modulate telomere length. Such reagents will have therapeutic use to limit the life span of tumor cells and to boost the proliferative potential of desired cell populations.
Hybridization, Parthenogenesis and Ploidy Changes in Vertebrates
Approximately 80 taxa of unisexual vertebrates have been described, including all-female species of whiptail lizards first reported in the early 1960s. A few years later allozyme analysis demonstrated that a parthenogenetic species harbored the genetic information from two sexual species that had hybridized to produce it. Upon reviewing this work four decades later, I was surprised that two key questions remained unanswered: (1) How are mature eggs are produced that carry the full complement of chromosomes in the absence of fertilization? (2) How is heterozygosity maintained over the course of many generations?
Our first set of experiments examined the DNA content in pre-meiotic oocytes from parthenogenetic and closely related sexual species and provided conclusive evidence that meiosis initiates with twice the number of chromosomes in the parthogenetic species (Figure 2A, B). By doubling the number of chromosomes prior to meiosis, the normal meiotic program produces diploid rather than haploid eggs. We next developed fluorescent in situ hybridization probes to distinguish between homologous chromosomes and demonstrated that pairing and recombination occur between genetically identical "sister chromosomes" instead of homologs (Figure 2C). This important deviation from the normal meiotic program explained the long-term maintenance of heterozygosity, which forms the basis of these lizards' ability to reproduce clonally and to compete with sexually reproducing species over many generations.
This initial foray into studying parthenogenesis further raised my interest in the underlying issues. True parthenogenesis avoids the "twofold cost of sex" by making no investment in males and by enabling each individual to produce offspring independently. Consequently, a single animal is theoretically capable of establishing its species in a new habitat. Why sexual reproduction is nevertheless so prevalent in nature has been referred to as the "queen of questions in evolutionary biology" by Graham Bell and has sparked a lively debate about the advantages of sex.
Interspecific hybridization endows clonal lineages with high levels of heterozygosity and plays a key role in the transition from sexual to parthenogenetic reproduction in vertebrates. Ploidy elevation further increases heterozygosity because the additional genome expands the genetic repertoire, which may permit adaptation to a changing environment or the exploitation of new ecological niches. Indeed, two-thirds of parthenogenetic species of whiptail lizards are triploids, whereas most of the diploid intermediates are believed to be extinct. However, hybridization events that superimpose a haploid sperm on a parthenogenetic egg and establish a new lineage with elevated ploidy had not been observed previously. Taking advantage of our laboratory colony, we have produced hybrid lizards, some of which have founded new genetically isolated lineages with elevated ploidy. The availability of new clonally reproducing lineages, as well as hybrids with impaired gonads, has opened up a new angle for investigating the molecular basis of parthenogenetic reproduction and the causes of hybrid sterility.
This research is supported in part by the Stowers Institute for Medical Research.
As of March 11, 2016