The nucleolus is primarily described as the control center for ribonuclear protein assembly and rRNA synthesis. However, there is increasing evidence that the nucleolus also plays a role in protein quality, genome stability and cell cycle progression. Previous studies demonstrated that the B-box type zinc finger protein ncl- 1 is tightly linked to nucleolar function, and that its loss leads to increased levels of the nucleolar marker fibrillarin (fib-1) and to an increased nucleolar area. These nucleolar phenotypes are accompanied by the abolishment of lifespan extension in the major longevity pathways. However, the molecular connection between ncl-1, nucleolar function and longevity remains elusive.
To identify molecular players and pathways that mediate the function of ncl-1 on longevity, I performed transcriptomic and proteomic analysis, comparing wild-type worms to ncl-1 worms in normal as well as the long-lived glp-1 background. This analysis revealed some shared and some background-specific regulation of distinct biological pathways upon loss of ncl-1: While nuclear outputs such as ribosome biogenesis and rRNA production are increased in multiple tested genotypes, proteasomal components are decreased in ncl-1 single mutants, while lysosomal components show lower abundance in glp-1;ncl-1. Interestingly, NCL-1 seems to affect at least a portion of regulated genes through direct binding of respective mRNAs. Based on transcriptomic and proteomic data, I conducted a follow-up RNA interference screen of potential mediators and uncovered nucleolar downstream processes of ncl- 1 including the RNase P/MRP complex and mitochondrial translation. Knockdown of the shared RNase P/MRP component POPL-1 as well as other RNase P/MRP factors extended lifespan in glp-1;ncl-1 mutants. The same was observed for other rRNA processing and ribosome assembly factors as well as proteins involved in mitochondrial translation such as MRPS-16. Reduction of popl-1 and mrps-16 extended lifespan independent of nucleolar size, fib-1 mRNA levels or steady state rRNA levels, thereby uncoupling nucleolar size from lifespan for the first time. Also, overall translation rate seems unaffected. Thus, the effect of NCL-1 on lifespan may
be mediated through altered ribosome assembly in glp-1 worms, while being unaffected in the N2 background.
To further investigate the molecular mechanism of NCL-1 action, I conducted a Yeast- 2-Hybrid assay and pulldown experiments to identify NCL-1 protein interactors. In a mini RNAi screen, I found that a reduction of the potential NCL-1 interactor and proteasomal factor RPN-11 affects nucleolar size in glp-1 mutants, indicating a direct connection of NCL-1 to the proteasome. In line with this, ncl-1 affects cellular proteostasis with the proteasome as a presumable key player, as ncl-1 mutants are short-lived under modest thermal stress and are less motile. In addition, I observed that overall ubiquitinylation levels are increased, and that proteasome substrates accumulate in ncl-1 mutants.
In the pulldown experiments with NCL-1, I identified several mitochondrial factors in the co-enriched fraction of NCL-1. I also found that NCL-1 forms round network-like structures and strong foci resembling mitochondria within the hypodermis and muscle, further supporting a mitochondrial connection.
Further on I established a connection between the RNAi machinery, nucleolar size and lifespan with a central focus on the argonaut protein NRDE-3 being required for longevity and affecting nucleolar size.
Using a range of genetic and biochemical approaches, I found that ncl-1 is a multifaceted gene that connects multiple important cellular pathways with a focus on nucleolar outputs, but also proteolytic processes. I identified the RNase P/MRP complex and mitochondrial translation as potential key processes for mediating the function of ncl-1 in longevity
