polysome-purification-from-soybean-symbiotic-nodules

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Instituto de Investigaciones Biológicas Clemente Estable

University of Virginia

Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.

Commitment of adult stem cells involves the activation of specific gene networks regulated from transcription to protein synthesis. Here, we used ribosome profiling to identify mRNAs regulated at the translational level, through both differential association to polysomes and modulation of their translational rates. We observed that translational regulation during the differentiation of human adipose-derived stromal cells (hASCs, also known as adipose-derived mesenchymal stem cells), a subset of which are stem cells, to adipocytes was a major regulatory event. hASCs showed a significant reduction of whole protein synthesis after adipogenic induction and a downregulation of the expression and translational efficiency of ribosomal proteins. Additionally, focal adhesion and cytoskeletal proteins were downregulated at the translational level. This negative regulation of the essential biological functions of hASCs resulted in a reduction in cell size and the potential of hASCs to migrate. We analyzed whether the inactivation of key translation initiation factors was involved in this observed major repression of translation. We showed that there was an increase in the hypo phosphorylated forms of 4E-BP1, a negative regulator of translation, during early adipogenesis. Our results showed that extensive translational regulation occurred during the early stage of the adipogenic differentiation of hASCs.

Downregulation of the protein synthesis machinery is a major regulatory event during early adipogenic differentiation of human adipose-derived stromal cells.

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control.

Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control.

Protein translation is a key step in gene expression. The development of Ribosome Profiling has allowed the global analysis of this process at sub-codon resolution. In the last years the method has been applied to several models ranging from bacteria to mammalian cells yielding a surprising amount of insight on the mechanism and the regulation of translation. In this review we describe the key aspects of the experimental protocol and comment on the main conclusions raised in different models.

Following Ribosome Footprints to Understand Translation at a Genome Wide Level.

Differences in social status are often mediated by agonistic encounters between competitors. Robust literature has examined social status-dependent brain gene expression profiles across vertebrates, yet social status and reproductive state are often confounded. It has therefore been challenging to identify the neuromolecular mechanisms underlying social status independent of reproductive state. Weakly electric fish, Gymnotus omarorum, display territorial aggression and social dominance independent of reproductive state. We use wild-derived G. omarorum males to conduct a transcriptomic analysis of non-breeding social dominance relationships. After allowing paired rivals to establish a dominance hierarchy, we profiled the transcriptomes of brain sections containing the preoptic area (region involved in regulating aggressive behaviour) in dominant and subordinate individuals. We identified 16 differentially expressed genes (FDR < 0.05) and numerous genes that co-varied with behavioural traits. We also compared our results with previous reports of differential gene expression in other teleost species. Overall, our study establishes G. omarorum as a powerful model system for understanding the neuromolecular bases of social status independent of reproductive state.

Brain transcriptomics of agonistic behaviour in the weakly electric fish Gymnotus omarorum, a wild teleost model of non-breeding aggression.

Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.

PDCD4 regulates axonal growth by translational repression of neurite growth-related genes and is modulated during nerve injury responses.

Functional genomics studies through transcriptomics, translatomics and proteomics have become increasingly important tools to understand the molecular basis of biological systems in the last decade. In most cases, when these approaches are applied to the nervous system, they are centered in cell bodies or somatodendritic compartments, as these are easier to isolate and, at least , contain most of the mRNA and proteins present in all neuronal compartments. However, key functional processes and many neuronal disorders are initiated by changes occurring far away from cell bodies, particularly in axons (axopathologies) and synapses (synaptopathies). Both neuronal compartments contain specific RNAs and proteins, which are known to vary depending on their anatomical distribution, developmental stage and function, and thus form the complex network of molecular pathways required for neuron connectivity. Modifications in these components due to metabolic, environmental, and/or genetic issues could trigger or exacerbate a neuronal disease. For this reason, detailed profiling and functional understanding of the precise changes in these compartments may thus yield new insights into the still intractable molecular basis of most neuronal disorders. In the case of synaptic dysfunctions or synaptopathies, they contribute to dozens of diseases in the human brain including neurodevelopmental (i.e., autism, Down syndrome, and epilepsy) as well as neurodegenerative disorders (i.e., Alzheimer's and Parkinson's diseases). Histological, biochemical, cellular, and general molecular biology techniques have been key in understanding these pathologies. Now, the growing number of omics approaches can add significant extra information at a high and wide resolution level and, used effectively, can lead to novel and insightful interpretations of the biological processes at play. This review describes current approaches that use transcriptomics, translatomics and proteomic related methods to analyze the axon and presynaptic elements, focusing on the relationship that axon and synapses have with neurodegenerative diseases.

Functional Genomics of Axons and Synapses to Understand Neurodegenerative Diseases.

Defining cellular mechanisms that drive Alzheimer's disease (AD) pathogenesis and progression will be aided by studies defining how gene expression patterns change during pre-symptomatic AD and ensuing periods of declining cognition. Previous studies have emphasized changes in transcriptome, but not translatome regulation, leaving the ultimate results of gene expression alterations relatively unexplored in the context of AD.

Article	Year
Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi. BMC genomics\| PubMed ID: 26054634	2015
Downregulation of the protein synthesis machinery is a major regulatory event during early adipogenic differentiation of human adipose-derived stromal cells. Stem cell research\| PubMed ID: 29156375	2017
Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control. PloS one\| PubMed ID: 29182646	2017
Following Ribosome Footprints to Understand Translation at a Genome Wide Level. Computational and structural biotechnology journal\| PubMed ID: 30069283	2018
Brain transcriptomics of agonistic behaviour in the weakly electric fish Gymnotus omarorum, a wild teleost model of non-breeding aggression. Scientific reports\| PubMed ID: 32528029	2020
PDCD4 regulates axonal growth by translational repression of neurite growth-related genes and is modulated during nerve injury responses. RNA (New York, N.Y.)\| PubMed ID: 32747606	2020
Functional Genomics of Axons and Synapses to Understand Neurodegenerative Diseases. Frontiers in cellular neuroscience\| PubMed ID: 34248504	2021
Transcriptome and Translatome Regulation of Pathogenesis in Alzheimer's Disease Model Mice. Journal of Alzheimer's disease : JAD\| PubMed ID: 35034904	2022

Guillermo Eastman

Biography

JoVE Journal Publications

Other Publications