Periodic Reporting for period 1 - NextGen RiBiomics (Next Generation Proteomic Analysis of Pre-Ribosomal Proteome Dynamics Coupled to Glucose Metabolism in Caner Cells)
Reporting period: 2016-04-01 to 2018-03-31
This project will focus on the relationship between the process of ribosome biogenesis (RiBi) and glucose metabolism in human cells. Solid tumour cells are continuously exposed to glucose deprivation and hypoxia microenvironments because of the inadequate vascular supply. Cancer cells in these tumours have to limit energy expenditure to survive under such energy-deprived conditions. Meanwhile, the most energy-consuming process in eukaryotic cells is protein synthesis by the ribosome and ribosome subunit biogenesis, so these steps are tightly regulated in response to metabolic changes. Therefore, characterising the mechanisms involved in these processes may lead to novel cancer treatment strategies, for example inducing deregulation of either protein synthesis, or RiBi, from metabolic control.
The ribosome, which consists of four ribosomal RNAs (rRNAs) and ∼80 ribosomal proteins (RPs), is essential for protein synthesis in the cell. The mammalian ribosome is composed of a large (60S) subunit and a small (40S) subunit. Its biogenesis takes place in a dedicated subnuclear compartment, the nucleolus, where a transcribed rRNA precursor (47S pre-rRNA in human) is assembled with proteins to form a large 90S pre-ribosomal particle (PR). Then the 47S pre-rRNA is processed to separate the 90S particle into pre-40S and pre-60S particles. These separated particles independently undergo maturation steps further and move from the nucleolus to the nucleoplasm then eventually to the cytoplasm.
However, the detailed molecular mechanisms involved in RiBi in human cells has not been fully elucidated. Moreover, the proteins involved in this process have also not been fully identified. A major reason for this is that the isolation of PRs from human cells is very challenging. Several biochemical subcellular fractionation steps, coupled with sucrose density gradient centrifugation (SDGC), is required. In contrast, a simpler co-immunoprecipitation approach allows efficient extraction of PRs from yeast cells, but this is not effective for analysis of extracts from human cells. Particularly, SDGC, which is the most widely used method for the isolation of ribosomes and PRs since the ribosome was discovered in 1960s, is widely recognized to have limitations, including limited reproducibility and the relatively long time required to set up and perform the analysis.
Therefore, a first major aim for this project was to establish a new, more efficient method for isolating ribosomes and PR complexes. Next, we aimed to optimise this new method for isolating PRs and to identify their components using Mass Spectrometry (MS)-based proteomic analysis. Finally, the aim was to compare the components of PRs extracted either from normal cells, or from glucose-starved cells, to try to identify proteins which play a role in the regulation of RiBi under conditions of glucose starvation.
The ribosome, which consists of four ribosomal RNAs (rRNAs) and ∼80 ribosomal proteins (RPs), is essential for protein synthesis in the cell. The mammalian ribosome is composed of a large (60S) subunit and a small (40S) subunit. Its biogenesis takes place in a dedicated subnuclear compartment, the nucleolus, where a transcribed rRNA precursor (47S pre-rRNA in human) is assembled with proteins to form a large 90S pre-ribosomal particle (PR). Then the 47S pre-rRNA is processed to separate the 90S particle into pre-40S and pre-60S particles. These separated particles independently undergo maturation steps further and move from the nucleolus to the nucleoplasm then eventually to the cytoplasm.
However, the detailed molecular mechanisms involved in RiBi in human cells has not been fully elucidated. Moreover, the proteins involved in this process have also not been fully identified. A major reason for this is that the isolation of PRs from human cells is very challenging. Several biochemical subcellular fractionation steps, coupled with sucrose density gradient centrifugation (SDGC), is required. In contrast, a simpler co-immunoprecipitation approach allows efficient extraction of PRs from yeast cells, but this is not effective for analysis of extracts from human cells. Particularly, SDGC, which is the most widely used method for the isolation of ribosomes and PRs since the ribosome was discovered in 1960s, is widely recognized to have limitations, including limited reproducibility and the relatively long time required to set up and perform the analysis.
Therefore, a first major aim for this project was to establish a new, more efficient method for isolating ribosomes and PR complexes. Next, we aimed to optimise this new method for isolating PRs and to identify their components using Mass Spectrometry (MS)-based proteomic analysis. Finally, the aim was to compare the components of PRs extracted either from normal cells, or from glucose-starved cells, to try to identify proteins which play a role in the regulation of RiBi under conditions of glucose starvation.
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.
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.
We have successfully developed a powerful new method – ‘Ribo Mega-SEC’ and shown that this offers major improvements over conventional approaches (such as SDGC), for analyzing translation-active polysomes and ribosome complexes. Our new method is rapid, convenient and provides highly reproducible fractionation. The technical limitations of previous methods for polysome isolation and analysis has hindered many researchers outside the field directly studying translation and ribosomes from analysing gene expression at the level of polysomes. Ribo Mega-SEC can therefore enable a much wider number of researchers interested in gene regulation in many different areas of biology to now benefit from including polysome analyses in their projects. Considering that previously there has been essentially no alternative to the laborious SDGC method for the analysis of polysomes/ribosomes and PRs, we believe that our new Ribo Mega-SEC approach is valuable and likely to have major impact.