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      System Wide Analyses have Underestimated Protein Abundances and Transcriptional Importance in Mammals

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          Abstract

          System wide surveys in mammals suggest that the protein expressed at the median abundance is present at 8,000 - 16,000 molecules per cell and that differences in mRNA expression between genes explain only 10-40% of the differences in protein levels. We find, however, that these surveys have significantly underestimated protein abundances. Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al., we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than the uncorrected protein data. To estimate the degree to which mRNA expression levels determine protein levels, it is critical to measure the error in protein and mRNA abundance data and to consider all genes. By taking direct measurements of experimental error into account, we estimate that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. By in addition modeling all genes' expression, we show that mRNA levels can explain at least 65% of protein levels for expressed genes and 100% for genes that are not expressed. We also employ a second strategy to determine the contribution of mRNA levels to protein expression. This shows that the median variance in translation rates directly measured by ribosome profiling in three human and mouse cell lines is 4.6 fold less than the variance inferred by Schwanhausser et al. and that based on this mRNA levels are expected to explain ~75% of the variance in expressed protein levels for the 4,212 detected genes and ~82% for all expressed genes. While the magnitude of our differently derived estimates vary, all suggest that the previous studies have significantly underestimated the importance of transcription.

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          Global quantification of mammalian gene expression control.

          Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.
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            Mammalian microRNAs predominantly act to decrease target mRNA levels

            Summary MicroRNAs (miRNAs) are endogenous ∼22-nucleotide RNAs that play important gene-regulatory roles by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (≥84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.
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              The impact of microRNAs on protein output.

              MicroRNAs are endogenous approximately 23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output.
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                Genetics

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