Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology.

Prokaryotes utilize polycistronic messages (operons) to co-translate proteins involved in the same biological processes. Whether eukaryotes achieve similar regulation by selectively assembling and translating monocistronic messages derived from different chromosomes is unknown. We employed transcript-specific RNA pulldowns and RNA-seq/RT-PCR to identify yeast mRNAs that co-precipitate as ribonucleoprotein (RNP) complexes. Consistent with the hypothesis of eukaryotic RNA operons, mRNAs encoding components of the mating pathway, heat shock proteins, and mitochondrial outer membrane proteins multiplex in trans, forming discrete messenger ribonucleoprotein (mRNP) complexes (called transperons). Chromatin capture and allele tagging experiments reveal that genes encoding multiplexed mRNAs physically interact; thus, RNA assembly may result from co-regulated gene expression. Transperon assembly and function depends upon histone H4, and its depletion leads to defects in RNA multiplexing, decreased pheromone responsiveness and mating, and increased heat shock sensitivity. We propose that intergenic associations and non-canonical histone H4 functions contribute to transperon formation in eukaryotic cells and regulate cell physiology.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

Evolthon: A community endeavor to evolve lab evolution.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.

Localizing mRNAs Encoding Mitochondrial Proteins in Yeast by Fluorescence Microscopy and Subcellular Fractionation.

Mitochondria are thought to have evolved from ancestral proteobacteria and, as a result of symbiosis, became an indispensable organelle in all eukaryotic cells. Mitochondria perform essential functions that provide the cell with ATP, amino acids, phospholipids, and both heme and iron-sulfur clusters. However, only 1% of mitochondrial proteins are encoded by the mitochondrial genome, while the remaining 99% are encoded in the nucleus. This raises a logistical challenge to the cell, as these nuclear-encoded proteins have to be translated, delivered to the mitochondrial surface, and translocated to its various compartments. Over the past decade, it was shown that subsets of mRNAs encoding mitochondrial proteins (mMPs) are localized to the mitochondrial surface in both yeast and mammalian cells. Moreover, factors (e.g., RNA-binding proteins) have been discovered that facilitate mMP targeting, and their loss leads to RNA mislocalization and defects in mitochondrial function (e.g., deficient respiration). Therefore, there is a demand in the field of mitochondrial biology to accurately measure mMP localization to the mitochondrial surface. In this chapter, we describe two techniques that allow for the visualization of mMPs using single-molecule fluorescent in situ hybridization and preparation of a highly enriched mitochondrial fraction followed by quantitative real-time PCR. Together, these techniques constitute powerful tools to link changes in mMP trafficking to defects in mitochondrial physiology.

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins.

Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to "MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system".

The MS2 system has been extensively used to visualize single mRNA molecules in live cells and follow their localization and behavior. In their Letter to the Editor recently published, Garcia and Parker suggest that use of the MS2 system may yield erroneous mRNA localization results due to the accumulation of 3' decay products. Here we cite published works and provide new data which demonstrate that this is not a phenomenon general to endogenously expressed MS2-tagged transcripts, and that some of the results obtained in their study could have arisen from artifacts of gene expression.