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Dosage and temporal thresholds in microRNA proteomics.

ABSTRACT: MicroRNAs (miRNAs) modulate protein and mRNA expression through translational repression and/or mRNA decay. In this study, we combined SILAC-based proteomics and RNAseq to identify primary targets based on measurements of protein and mRNA repression and analysis of transcript 3'UTR sequences. The primary target set was used to compare different prediction algorithms, revealing higher stringency of selection by Targetscan and PITA compared with miRanda, at the expense of higher false negatives. A key finding was that significant and unexpected variations occurred in the kinetics of repression as well as the sensitivity to exogeneous miRNA concentration. Bimodal thresholds were observed, which distinguished responses to low (10 nm) versus high (50-100 nm) miRNA, as well as the onset of repression at early (12-18 h) versus late (36-48 h) times. Similar behavior was seen at the transcript level with respect to kinetics of repression. The differential thresholds were most strongly correlated with ??G, the net free energy of miRNA-target interactions, which mainly reflected inverse correlations with ?Gopen, the free energy of forming 3'UTR secondary structures, at or nearby the miRNA seed matching sites. Thus, our working model is that protein binding or other competitive mechanisms variably interfere with the accessibility of miRISC to the transcript binding site. In addition, biphasic responses were observed in a subset of proteins that were partially down-regulated at early times, and further down-regulated at later times. Taken together, our findings provide evidence for varying modes of miRNA target repression, which lead to different thresholds of target responses with respect to kinetics and concentration, and predict that certain transcripts will show graded responses in sensitivity and fold-change under cellular conditions that lead to varying steady state miRNA levels.

SUBMITTER: Lee T

PROVIDER: S-EPMC4350026 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Dosage and temporal thresholds in microRNA proteomics.

Lee Thomas T Wang Nan N Houel Stephane S Couts Kasey K Old William W Ahn Natalie N

Molecular & cellular proteomics : MCP 20141202 2

MicroRNAs (miRNAs) modulate protein and mRNA expression through translational repression and/or mRNA decay. In this study, we combined SILAC-based proteomics and RNAseq to identify primary targets based on measurements of protein and mRNA repression and analysis of transcript 3'UTR sequences. The primary target set was used to compare different prediction algorithms, revealing higher stringency of selection by Targetscan and PITA compared with miRanda, at the expense of higher false negatives. A ...[more]

PMID: 25467838

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Project description:Background:Increasing attention is payed to the contribution of somatosensory processing in motor control. In particular, temporal somatosensory discrimination has been found to be altered differentially in common movement disorders. To date, there have only been speculations as to how impaired temporal discrimination and clinical motor signs may relate to each other. Prior to disentangling this relationship, potential confounders of temporal discrimination, in particular age and peripheral nerve conduction, should be assessed, and a quantifiable measure of proprioceptive performance should be established. Objective:To assess the influence of age and polyneuropathy (PNP) on somatosensory temporal discrimination threshold (STDT), temporal discrimination movement threshold (TDMT), and behavioral measures of proprioception of upper and lower limbs. Methods:STDT and TDMT were assessed in 79 subjects (54 healthy, 25 with PNP; age 30-79 years). STDT was tested with surface electrodes over the thenar or dorsal foot region. TDMT was probed with needle electrodes in flexor carpi radialis (FCR) and tibialis anterior (TA) muscle. Goniometer-based devices were used to assess limb proprioception during (i) active pointing to LED markers, (ii) active movements in response to variable visual cues, and (iii) estimation of limb position following passive movements. Pointing (or estimation) error was taken as a measure of proprioceptive performance. Results:In healthy subjects, higher age was associated with higher STDT and TDMT at upper and lower extremities, while age did not correlate with proprioceptive performance. Patients with PNP showed higher STDT and TDMT values and decreased proprioceptive performance in active pointing tasks compared to matched healthy subjects. As an additional finding, there was a significant correlation between performance in active pointing tasks and temporal discrimination thresholds. Conclusion:Given their notable impact on measures of temporal discrimination, age and peripheral nerve conduction need to be accounted for if STDT and TDMT are applied in patients with movement disorders. As a side observation, the correlation between measures of proprioception and temporal discrimination may prompt further studies on the presumptive link between these two domains.

Project description:The ability to derive neural progenitors, differentiated neurons and glial cells from human embryonic stem cells (hESCs) with high efficiency holds promise for a number of clinical applications. However, investigating the temporal events is crucial for defining the underlying mechanisms that drive this process of differentiation along different lineages. We carried out quantitative proteomic profiling using a multiplexed approach capable of analyzing eight different samples simultaneously to monitor the temporal dynamics of protein abundance as human embryonic stem cells differentiate into motor neurons or astrocytes. With this approach, a catalog of approximately 1200 proteins along with their relative quantitative expression patterns was generated. The differential expression of the large majority of these proteins has not previously been reported or studied in the context of neural differentiation. As expected, two of the widely used markers of pluripotency, alkaline phosphatase (ALPL) and LIN28, were found to be downregulated during differentiation, while S-100 and tenascin C were upregulated in astrocytes. Neurofilament 3 protein, doublecortin and CAM kinase-like 1 and nestin proteins were upregulated during motor neuron differentiation. We identified a number of proteins whose expression was largely confined to specific cell types, embryonic stem cells, embryoid bodies and differentiating motor neurons. For example, glycogen phosphorylase (PYGL) and fatty acid binding protein 5 (FABP5) were enriched in ESCs, while beta spectrin (SPTBN5) was highly expressed in embryoid bodies. Karyopherin, heat shock 27 kDa protein 1 and cellular retinoic acid binding protein 2 (CRABP2) were upregulated in differentiating motor neurons but were downregulated in mature motor neurons. We validated some of the novel markers of the differentiation process using immunoblotting and immunocytochemical labeling. To our knowledge, this is the first large-scale temporal proteomic profiling of human stem cell differentiation into neural cell types highlighting proteins with limited or undefined roles in neural fate.

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Dosage and temporal thresholds in microRNA proteomics.

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Dosage and temporal thresholds in microRNA proteomics.

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