The multifunctional exoribonuclease protein PNPase is implicated as a potential target for cancer therapy as well as causing mitochondrial disorders in humans, but there has yet to be a whole animal knockdown model created. In this study, C. elegans was used to investigate the effect of knocking down pnpt-1, the gene that encodes PNPase. It was discovered that pnpt-1 knockdown significantly extends lifespan via an increase in superoxide production similar to other known mitochondrial lifespan extension pathways. Additionally, mitochondrial networks, size and respiration are affected indication of other mitochondrial dysfunction.. PNPase is also known to transport small RNAs into the mitochondria which in turn can affect mitochondria RNA splicing and translation of proteins involved in respiration. Further investigation showed a significant accumulation of polycistronic mitochondrial transcripts in knockdown animals. Lastly, this model has shown that PNPase knockdown is functionally comparable across species and is a viable model for future studies.

PNPase, polynucleotide phosphorylase, is a multifunctional exoribonuclease protein with 3` terminal oligonucleotide polymerase activity. Coded by the PNPT1 gene, the protein is associated with mitochondrial homeostasis and functions as a possible target for cancer therapy. In this study, C. elegans was used to investigate the effect of mutation and overexpression of pnpt-1, the gene that encodes PNPase. It was determined that two specific mutations in pnpt-1 did not affect PNPase expression nor did they produce deleterious phenotypes that affected polycistronic transcript accumulation or ROS production. Creation of a stable overexpression model was achieved through Fusion PCR. However, different transgenic strains overexpressing PNPase produced opposite results for polycistronic transcript accumulation while ROS production saw no significant change, suggesting a mosaic overexpression model. In a cancer model, exogenous PNPase was present in the pachytene region of the germline and where expressed the cells were in non-germline cells suggesting differentiation mechanisms associated with overexpression of PNPase. However, further analysis of different mutations in pnpt-1 or optimizations to the overexpression model are necessary to provide a better understanding of PNPase function with mitochondria homeostasis and in a cancer model setting.

Upon termination of translation, the fate of ribosomes is determined largely by the rate at which cells are growing. During periods of exponential growth, ribosomes are rapidly recycled, translation is re-initiated, and the ribosomes are extremely stable. However, when nutrient sources become limiting, and ribosomes are not actively translating, they may become substrates for degradation. While this phenomenon is well known, details of how the process is initiated and what are the signals for degradation have, until now, remained elusive. Here, I present in vitro and in vivo data showing that free ribosome subunits are the targets of degradative enzymes, whereas 70S particles that remain associated are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation. Thus, the simple presence of free 50S and 30S subunits is sufficient to serve as the mechanism that initiates ribosome degradation. In order to identify RNases involved in ribosome degradation, both in vitro and in vivo assays were developed. Together, they have provided evidence for a multi-step degradation process involving both endo- and exoribonucleases. Examination of extracts from strains deficient in known RNases revealed that the endoribonucleases, RNase E and RNase G, may be involved in the initial cleavages. The resulting fragments, some of which are small enough oligoribonucleotides that they remain part of the acid-soluble fraction are degraded to mononucleotides primarily by the 3'-5' exoribonucleases, RNase R and polynucleotide phosphorylase.

PNPase is a gene implicated as a potential target for cancer therapy; human mutations also present with deafness, myopathies, and neuropathies. In this study, C. elegans was used to investigate the effect of knocking out PNPase in a whole animal. C. elegans knockdown studies have reported an extended lifespan via an increase in ROS production. Further noted are larger mitochondria and an increase in fzo-1 expression. Knockout animals previously constructed using CRISPR/Cas9 were used for this study. We aimed to confirm these findings validating previous studies. It was discovered that PNPase knockout animals demonstrated a similar lifespan extension that was resolved with the addition of antioxidants in the media. All subsequent findings contradicted those of the knockdown studies. Resequencing of knockout animals demonstrated no existing mutation and studies were discontinued. New mutants will advance future analyses and validate prior investigations.