To establish a new approach for fractionation of ribosomes, we decided to employ a Size Exclusion Chromatography (SEC)-based ultra High Pressure Liquid Chromatography (uHPLC) method. Arguably, this provides the most efficient and reproducible method for fractionation-based biochemical analysis. We also decided to fractionate cytoplasmic ribosomes and polysomes first, as these are more easily extracted from cells, as compared to PRs in the nucleolus.
We first optimized the method for preparation of lysates from cells. Next, we optimized both the choice of pore size for the SEC column and the flow rate. We compared the SEC chromatograms using three different pore size SEC columns (300, 1,000, or 2,000 Å). This showed that only the largest pore size SEC column successfully resolved complexes in the polysome size range. We then assigned the resulting peaks by injecting either ribosomal subunits, or polysomes isolated by SDGC, onto the SEC column. We next optimized the column flow rate and showed that the polysome and ribosomal subunits could be fractionated successfully by SEC in as little as 15 min, which contrasts with the many hours required for SDGC separation.
We characterized the SEC profiles further, using both western and northern blotting to compare the distribution of RPs, rRNAs, and mRNAs across the SEC fractions. We also examined the stability and activity of polysomes, isolated by SEC, by re-analyzing the isolated polysome fractions by a second round of fractionation using SDGC and also by using electron microscopy. We detected no dissociated ribosomal subunits from SEC purified polysome fractions, showing that the isolated complexes are stable. Moreover, the translation complexes isolated from the SEC polysome fractions show peptidyl transferase activity.
We have carefully evaluated the reproducibility of polysome fractionation using the SEC-based approach. This showed very high Pearson correlation coefficients (~0.99) across multiple biological and technical replicates. In comparison, polysome fractionation by SDGC showed consistently lower Pearson correlation coefficients than those measured from SEC. Moreover, the largest differences between separate SDGC replicates were observed in the polysome region.
Having characterized and optimized the uHPLC-based method for efficient and reproducible isolation of polysomes from either cell lines or tissues, we have initiated experiments using this approach to study how metabolism and stress changes the relative level of polysomes and ribosomal subunits in mouse liver tissues. This work is yielding promising results and the project is ongoing.
In summary, we have successfully developed an efficient new SEC-based method that provides a major improvement over previous approaches for isolating polysomes and ribosome subunits. We have termed this new method “Ribo Mega-SEC” and a manuscript describing this method submitted to eLife is currently under revision, having been favorably reviewed.
We have subsequently proceeded to optimize the SEC method also for isolation of PRs. This required a change in the composition of the running buffer to create conditions suitable for the efficient separation of PRs. We compared separation profiles using two different SEC columns (1,000 or 2,000 Å). This showed that the 2,000 Å SEC column successfully resolved three different PRs, i.e. 90S, pre-60S and pre-40S. We have collected these PRs across the fractions and prepared the samples for MS analyses to identify the protein components of the isolated PRs. This work is ongoing.
