measurement-protein-turnover-rates-senescent-non-dividing-cultured

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

National Institutes of Health

Senescence is a state of long-term cell cycle arrest that arises in cells that have incurred sublethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence-associated secretory phenotype (SASP). On the other hand, these progrowth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.

Translational Control during Cellular Senescence.

Just as eukaryotic circular RNA (circRNA) is a product of intracellular backsplicing, custom circRNA can be synthesized in vitro using a transcription template in which transposed halves of a split group I intron flank the sequence of the RNA to be circularized. Such permuted intron-exon (PIE) constructs have been used to produce circRNA versions of ribozymes, mimics of viral RNA motifs, a streptavidin aptamer, and protein expression vectors for genetic engineering and vaccine development. One limitation of this approach is the obligatory incorporation of small RNA segments (E1 and E2) into nascent circRNA at the site of end-joining. This restriction may preclude synthesis of small circRNA therapeutics and RNA nanoparticles that are sensitive to extraneous sequence, as well as larger circRNA mimics whose sequences must precisely match those of the native species on which they are modelled. In this work, we used serial mutagenesis and in vitro selection to determine how varying E1 and E2 sequences in a thymidylate synthase (td) group I intron PIE transcription template construct affects circRNA synthesis yield. Based on our collective findings, we present guidelines for the design of custom-tailored PIE transcription templates from which synthetic circRNAs of almost any sequence may be efficiently synthesized.

Matthew Payea

Laboratory of Genetics and Genomics,
National Institute on Aging Intramural Research Program,
Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program

National Institutes of Health

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Matthew Payea

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Genetics and Genomics,National Institute on Aging Intramural Research Program,Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program

National Institutes of Health

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Laboratory of Genetics and Genomics,
National Institute on Aging Intramural Research Program,
Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program