Polysome Profiling in <em>Leishmania</em>, Human Cells and Mouse Testis

Abstract

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.

Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations

“…In experiments with inhibition of transcription, cells were treated with 8 μM of actinomycin D (Sigma-Aldrich) for different periods of time as indicated. Artificial OmpA mRNA [52] was added to the samples of the actinomycin D experiment before total RNA purification to use it for normalization in RT-qPCR.…”

Mitofusin2 cooperates with Nuage-associated proteins and involves mRNA translational machinery in controlling mRNA fates during spermatogenesis

“…Testicular seminiferous tubules were dissected and collected according to previous literature with minor modifications(Aboulhouda et al, 2017; Karamysheva et al, 2018). Briefly after adding polysome extraction buffer (20mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail (EDTA-free), 200 μg/ml CHX, 200 units/mL of RNase inhibitor), the tubules were transferred to a small (0.5-1.0 mL) Dounce homogenizer to disrupt the tissues with seven to eight strokes of the glass pestle.…”

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

“…Polysome profiling experiments were completed as published earlier [25]. Briefly, for polysome profiling experiments, Leishmania promastigotes including WT, c14dm ̅ , c14dm ̅ /+C14DM were grown in flasks until cell density reached 5 x 10 6 cells/ml.…”

“…Cells were lysed on ice in a buffer containing 20 mM Hepes-KOH (pH 7.5), 10 mM MgCl2, 100 mM KCl, 2 mM DTT, 1% NP-40, 1 x protease inhibitor cocktail (EDTA-free) from Millipore Sigma, 200 units/ml RNasin (Thermo Fisher Scientific), and 100 µg/ml of cycloheximide. Details of cell lysate preparation are described previously [25]. The lysate was clarified by centrifugation at 11,200 g for 10 minutes at 4 °C.…”