Generation of ribosome imprinted polymers for sensitive detection of translational responses

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of “smart materials”, namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation

The global dynamics of RNA stability orchestrates responses to cellular activation

Transcriptomics is used to quantify changes in accumulated levels of mRNAs following cellular activation. These changes arise from the opposing fluxes of transcription and mRNA decay, both of which affect the functional dynamics of global gene expression. A study published recently in BMC Genomics focuses on the contribution made by mRNA stability in shaping the kinetics of gene responses in mammalian cells

Yeast Mitochondrial Biogenesis: A Role for the PUF RNA-Binding Protein Puf3p in mRNA Localization

The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes) have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes) have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators). Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings for the global study of mitochondria biogenesis

A Screen for RNA-Binding Proteins in Yeast Indicates Dual Functions for Many Enzymes

Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation

The influence of physical exercise on the generation of TGF-β1, PDGF-AA, and VEGF-A in adipose tissue

Adipose tissue is an important organ that produces and secretes hormones and cytokines, including TGF-β1, PDGF-AA, and VEGF-A. The goal of the present study was to investigate the influence of a single session of acute exercise, as well as the prolonged endurance training on the production of TGF-β1, PDGF-AA, and VEGF-A in the subcutaneous white adipose tissue in rats. Rats were randomly divided into two groups: untrained (UT, n = 30) and trained rats (T, subjected to 6-week endurance training with increasing load, n = 29). Both groups were subjected to an acute exercise session with the same work load. The rats were killed before (UTpre, Tpre), immediately after (UT0h, T0h), or 3 h (UT3h, T3h) after exercise and adipose tissue samples collected. Growth factor mRNA was evaluated using RT-PCR; the protein levels were measured before and after training (UTpre and Tpre) using the immunoenzymatic method. TGF-β1 and PDGF-AA mRNA levels were decreased in the UT3h rats compared to the UTpre rats (P = 0.0001 and P = 0.03, respectively), but the VEGF-A mRNA level remained unchanged in the UT0h and UT3h rats compared to UTpre rats. TGF-β1, PDGF-AA and VEGF-A mRNA levels were decreased in the T3h rats compared to Tpre (P = 0.0002, P = 0.02, and P = 0.03, respectively). TGF-β1, PDGF-AA and VEGF-A mRNA levels significantly increased in the Tpre rats compared to UTpre (all P = 0.0002). However, the protein levels remained constant. In conclusion, prolonged physical exercise increases growth factor mRNA in adipose tissue but not protein levels

Transcriptome Kinetics Is Governed by a Genome-Wide Coupling of mRNA Production and Degradation: A Role for RNA Pol II

Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II) subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism

Heat shock partially dissociates the overlapping modules of the yeast protein-protein interaction network: a systems level model of adaptation

Network analysis became a powerful tool in recent years. Heat shock is a well-characterized model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a "stratus-cumulus" type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes.Comment: 24 pages, 6 figures, 2 tables, 70 references + 22 pages 8 figures, 4 tables and 8 references in the enclosed Supplemen

Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed