Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.
Parkinson’s disease (PD) is a neurodegenerative disorder classified by the loss of dopaminergic neurons in the substantia nigra pars compacta, the region of the brain that is responsible for motor control. Surviving neurons in this region contain aggregated protein alpha-Synuclein (αSyn) in the form of cytoplasmic inclusions, referred to as Lewy bodies. Changes in αSyn expression are also associated with PD and its progression. Previously, we demonstrated that signal recognition particle (SRP) and Argonaute 2 (AGO2) proteins are involved in protein quality control at the ribosome during translation. We also demonstrated that SRP has an mRNA protection function in addition to a protein targeting function, thus controlling mRNA and protein expression. In this study, we tested involvement of these factors in αSyn biogenesis. We hypothesize that loss of these factors may interfere with αSyn expression, and subsequently, be associated with PD. Using depletion assays in human cell culture and analysis of these proteins in the brains of deceased PD patients, we demonstrate that SRP and AGO2 are involved in the control of αSyn expression and AGO2 has reduced expression in PD. We show for the first time that SRP is involved in mRNA protection of αSyn, a protein that does not have a signal sequence or transmembrane span. Our findings suggest that SRP may interact with a hydrophobic domain in the middle of αSyn during translation. Understanding the molecular mechanisms controlling αSyn biogenesis in cells is vital to developing preventative therapies against PD.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.