Ribosome profiling the cell cycle: lessons and challenges

Aramayo, Rodolfo; Polymenis, Michael

doi:10.1007/s00294-017-0698-3

Cited by 22 publications

(23 citation statements)

References 38 publications

(79 reference statements)

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“…We used centrifugal elutriation to obtain our synchronous cultures of 153 ribosomal protein paralog mutants. Unlike arrest-and-release synchronization approaches, 154 centrifugal elutriation maintains as much as possible the normal coupling of cell growth with cell 155 division (Aramayo and Polymenis, 2017;Soma et al, 2014). To collect enough cells for these 156 experiments, we followed the same approach as in our previous work on wild type cells (Blank 157 et al, 2017).…”

Section: Loss Of Rpl22ap Reduces Overall Protein Synthesis 133mentioning

confidence: 99%

Translational control of methionine and serine metabolic pathways underpin the paralog-specific phenotypes of Rpl22 ribosomal protein mutants in cell division and replicative longevity

Maitra

et al. 2020

Preprint

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21A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) 22 often display distinct phenotypes. Some may reflect general effects due to lower growth rate 23 and ribosome levels, but a number of diverse phenotypes cannot be explained through this 24 mechanism. Yeast and other organisms live longer when they lack specific ribosomal proteins, 25 especially of the large 60S subunit of the ribosome. However, longevity is neither associated 26 with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. 27Here, we comprehensively queried actively dividing RP paralog mutants through the cell cycle. 28Our data link transcriptional, translational, and metabolic changes to phenotypes associated 29 with the loss of paralogous RPs. We uncovered specific translational control of transcripts 30 encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) 31 pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites 32 associated with 1C metabolism. Loss of 1C enzymes, such as the serine 33 hydroxymethyltransferase Shm2p increased the longevity of wild type cells. These results 34 provide a molecular basis for paralog-specific phenotypes in ribosomal mutants and underscore 35 the significance of 1C metabolic pathways in mechanisms of cell division and cellular aging. 1C 36 pathways exist in all organisms, including humans, and targeting the relevant enzymes could 37 represent longevity interventions. 38Rpl22Ap does (He et al., 2014; Hoose et al., 2012;Truong et al., 2013). Dysregulation of 62 translation is also strongly linked with aging. The number of times a yeast cell can divide and 63 generate daughter cells defines its replicative lifespan (Steffen et al., 2009). Protein synthesis is 64 dysregulated in aged cells, and it is thought to be a driver of aging (Janssens et al., 2015). 65Mutations in ribosomal proteins of the large (60S) subunit constitute a significant class of pro-66 longevity mutations in yeast and other species (Kaeberlein and Kennedy, 2011; Kaeberlein et 67 al., 2005;McCormick et al., 2015;Steffen et al., 2008; Steffen et al., 2012). The rpl association 68 with longevity, however, is often paralog-specific and complex. For example, the Rpl22 double 69 paralog deletion is viable, but not long-lived (Steffen et al., 2012). The single rpl22aΔ mutants is 70 long-lived, but rpl22b∆ cells are not long-lived (Steffen et al., 2012). In other ribosomal proteins, 71 e.g., Rpl34, loss of either of the Rpl34 paralogs promotes longevity (Steffen et al., 2012). 72Importantly, bulk inhibition of translation with cycloheximide at various doses does not increase 73 lifespan (Steffen et al., 2008). The above observations argue that simple relations between 74 ribosome content, protein synthesis capacity, or generation time cannot sufficiently explain the 75 longevity of rpl paralog mutants. To account for these paralog-specific phenotypes, we decided 76 to identify patterns of translational ...

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Section: Loss Of Rpl22ap Reduces Overall Protein Synthesis 133mentioning

confidence: 99%

Translational control of methionine and serine metabolic pathways underpin the paralog-specific phenotypes of Rpl22 ribosomal protein mutants in cell division and replicative longevity

Maitra

et al. 2020

Preprint

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“…To apply genome-wide methods for the identification of cell cycle-dependent changes in the abundance of molecules of interest, one must first obtain highly synchronous cell cultures. Preferably, synchronization must be achieved in a way that minimally perturbs cellular physiology and the coordination between cell growth and division (Mitchison, 1971; Aramayo and Polymenis, 2017). When cells are chemically or genetically arrested in the cell cycle to induce synchrony, known arrest-related artifacts can bias the results (Mitchison, 1971; Ly et al ., 2015; Aramayo and Polymenis, 2017).…”

Section: Resultsmentioning

confidence: 99%

“…Preferably, synchronization must be achieved in a way that minimally perturbs cellular physiology and the coordination between cell growth and division (Mitchison, 1971; Aramayo and Polymenis, 2017). When cells are chemically or genetically arrested in the cell cycle to induce synchrony, known arrest-related artifacts can bias the results (Mitchison, 1971; Ly et al ., 2015; Aramayo and Polymenis, 2017). An alternative synchronization method is elutriation, a physical process that fractionates an asynchronous cell population by cell size and sedimentation density properties of the cells, with minimal perturbation of cellular functions (Lindahl, 1948; Creanor and Mitchison, 1979; Banfalvi, 2008).…”

Section: Resultsmentioning

confidence: 99%

Abundances of transcripts, proteins, and metabolites in the cell cycle of budding yeast reveals coordinate control of lipid metabolism

Blank

Papoulas

Maitra

et al. 2019

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Establishing the pattern of abundance of molecules of interest during cell division has been a long-standing goal of cell cycle studies. In several systems, including the budding yeast Saccharomyces cerevisiae, cell cycle-dependent changes in the transcriptome are well studied. In contrast, few studies queried the proteome during cell division, and they are often plagued by low agreement with each other and with previous transcriptomic datasets. There is also little information about dynamic changes in the levels of metabolites and lipids in the cell cycle. Here, for the first time in any system, we present experiment-matched datasets of the levels of RNAs, proteins, metabolites, and lipids from un-arrested, growing, and synchronously dividing yeast cells. Overall, transcript and protein levels were correlated, but specific processes that appeared to change at the RNA level (e.g., ribosome biogenesis), did not do so at the protein level, and vice versa. We also found no significant changes in codon usage or the ribosome content during the cell cycle. We describe an unexpected mitotic peak in the abundance of ergosterol and thiamine biosynthesis enzymes. Although the levels of several metabolites changed in the cell cycle, by far the most significant changes were in the lipid repertoire, with phospholipids and triglycerides peaking strongly late in the cell cycle. Our findings provide an integrated view of the abundance of biomolecules in the eukaryotic cell cycle and point to a coordinate mitotic control of lipid metabolism.

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“…[30] One challenge with these methods is that they require a large number of cells for each sample; therefore, an analysis of cell-cycle-dependent translation regulation must involve synchronization. [31] Polysome profiling showed that polysomes remain stable in mitosis when cells are synchronized by double thymidine block, although they are disrupted by nocodazole treatment, [32] again highlighting the impact of the choice of synchronization method. Translation during mitosis was found to be attenuated due to slower elongation, [32,33] which is consistent with the presence of maintained polysomes since polysome profiling cannot distinguish between unaltered active translation and slowed-down elongation.…”

Section: To Translate or What To Translate That Is The Questionmentioning

confidence: 99%

Cell‐Cycle‐Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches

Anda

Grallert

2019

BioEssays

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It is a long‐standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell‐cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges. Cell‐cycle‐dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress responses that, in turn, affect translation rates. Further, it is argued that previously reported cell‐cycle‐dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell‐cycle‐dependent manner. Novel techniques allowing a genome‐wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell‐cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased.

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Ribosome profiling the cell cycle: lessons and challenges

Cited by 22 publications

References 38 publications

Translational control of methionine and serine metabolic pathways underpin the paralog-specific phenotypes of Rpl22 ribosomal protein mutants in cell division and replicative longevity

Translational control of methionine and serine metabolic pathways underpin the paralog-specific phenotypes of Rpl22 ribosomal protein mutants in cell division and replicative longevity

Abundances of transcripts, proteins, and metabolites in the cell cycle of budding yeast reveals coordinate control of lipid metabolism

Cell‐Cycle‐Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches

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