Proteolysis-dependent regulation of telomerase

Eukaryotes maintain their genomes in the form of linear chromosomes. Because of the inability of the replication machinery to copy linear DNA molecules to the very end, the chromosomes were predicted to become shorter with every round of replication. This phenomenon was called “the end replication problem” by Watson and Olovnikov. Later, it has been discovered that eukaryotes have evolved telomeres, non-coding DNA repeats at the chromosomal ends, and telomerase, a ribonucleoprotein that extends the telomeres to overcome “the end replication problem”. Telomerase is known to be downregulated in humans through the development resulting in progressive telomere shortening and replicative senescence with age. This is believed to be one of the major tumour suppressor mechanisms. However, most cancer cells reactivate telomerase to become immortal. Thus, understating telomerase regulation is one of the central questions in the field of cancer biology. Telomerase complex formation involves several component maturation and assembly steps. In Saccharomyces cerevisiae, the steady-state levels of the catalytic subunit Est2 are significantly reduced when its interaction with the telomerase RNA component TLC1 is impaired. I have found that Est2 not bound to TLC1 undergoes degradation in a proteasome-dependent manner. Loss of Tom1, an E3-ubiquitin ligase, leads to an increase in the Est2 levels accompanied by a decrease in the Est2 degradation rate. Consistent with these findings, tom1 mutants have longer telomeres. Furthermore, Tom1 physically interacts with Est2, specifically through recognizing its RNA binding domain. Disruption of TOM1 does inhibit proteolysis of Est2 but does not stop it completely, suggesting the existence of an additional, Tom1-independent degradation pathway of Est2. Interestingly, the Est2 levels are reduced in cells grown at elevated temperatures. This decrease is caused by the temperature-triggered degradation of Est2. This degradation resembles proteolysis through the protein quality control system, however, the exact regulator of this mechanism is yet unknown. I propose that regulation of the telomerase through the proteolysis contributes to telomere length homeostasis indirectly through controlling the levels of Est2. The human Est2 homologue hTERT is also degraded in a proteasome-dependent manner. Thus, the proteolysis-dependent regulation of the telomerase might be evolutionarily conserved. RNA-free hTERT is believed to have extra-telomeric roles and this regulation might balance its telomeric and extra-telomeric functions

A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo

Abstract Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes