Abstract
Two novel approaches were recently suggested for genome-wide identification of protein aspects synthesized at a given time. Ribo-Seq is based on sequencing all the ribosome protected mRNA fragments in a cell, while PUNCH-P is based on mass-spectrometric analysis of only newly synthesized proteins. Here we describe the first Ribo-Seq/PUNCH-P comparison via the analysis of mammalian cells during the cell-cycle for detecting relevant differentially expressed genes between G1 and M phase. Our analyses suggest that the two approaches significantly overlap with each other. However, we demonstrate that there are biologically meaningful proteins/genes that can be detected to be post-transcriptionally regulated during the mammalian cell cycle only by each of the approaches, or their consolidation. Such gene sets are enriched with proteins known to be related to intra-cellular signalling pathways such as central cell cycle processes, central gene expression regulation processes, processes related to chromosome segregation, DNA damage, and replication, that are post-transcriptionally regulated during the mammalian cell cycle. Moreover, we show that combining the approaches better predicts steady state changes in protein abundance. The results reported here support the conjecture that for gaining a full post-transcriptional regulation picture one should integrate the two approaches.
Highlights
Two novel approaches were recently suggested for genome-wide identification of protein aspects synthesized at a given time
Ribo-Seq is based on the total number of ribosomes on the mRNA molecules related to a certain gene; PUNCH-P, on the other hand, is based on the total amount of nascent peptide emerging from the ribosomes on the mRNA molecules related to a certain gene which are translating at the time of the experiment
Our analyses demonstrate that the correlation between the G1 and M phases of steady state protein levels and Ribo-Seq (r(PSS,RP)) are: 0.70 (p < 10−454) and 0.70 (p < 10−454) respectively; the correlation is significant and high when controlling for mRNA levels (r (PSS,RP|mRNA): 0.45 (p = 2.4·10−252) and 0.47 (p = 4·10−280)
Summary
Two novel approaches were recently suggested for genome-wide identification of protein aspects synthesized at a given time. Ribo-Seq is based on sequencing all the ribosome protected mRNA fragments in a cell, while PUNCH-P is based on mass-spectrometric analysis of only newly synthesized proteins. We describe the first Ribo-Seq/PUNCH-P comparison via the analysis of mammalian cells during the cell-cycle for detecting relevant differentially expressed genes between G1 and M phase. We demonstrate that there are biologically meaningful proteins/genes that can be detected to be posttranscriptionally regulated during the mammalian cell cycle only by each of the approaches, or their consolidation. A new approach called PUNCH-P31,32 was proposed This approach is based on the combination of biotinylated puromycin with MS analysis to globally label newly synthesized proteins, enabling identifying the proteins translated in a certain condition. Since not all ribosomes on the mRNA (i.e. can be detected by Ribo-Seq) are translating[33,34,35] at a certain moment (i.e. can be detected by PUNCH-P), the signal detected by these approaches is not identical
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