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The proper timing of mRNA translation is crucial across many biological systems for processes such as intercellular communication, body pattern formation, and morphogenesis. The mainD. melanogasterposterior determinant,oskar, is maternally transcribed, but only translated when properly localized at the oocyte’s posterior cortex. Bruno 1 and Cup are two effector proteins known to participate in multiple aspects ofoskarmRNA regulation. Current model describes a mechanism in which Bruno 1 is necessary for Cup’s recruitment tooskarmRNA, and Bruno 1 is indispensable for its translational repression. Here, we reveal that the Bruno 1-Cup interaction, as well as their interdependent influence on each other’s mRNA and protein expression, lead to preciseoskarmRNA regulation during early oogenesis. We show that these factors stably associate with theoskarmRNAin vivo, but surprisingly, Bruno 1’s stable association withoskarmRNA depends on Cup, while Bruno 1 is not necessary for Cup association tooskarmRNA. During early oogenesis, Cup, not Bruno 1, is the essential factor foroskarmRNA repression. Cup is a crucial P-body member that maintains proper P-body morphology during oogenesis, as well as it is necessary foroskarmRNA’s association with P-bodies, thus driving the translational repression and stability ofoskarmRNA. Our experimental results collectively suggest a regulatory mechanism where a feedback loop between Bruno 1 and Cup coordinatesoskarmRNA regulation in the egg chamber allowing for proper development to occur.

The 2010 Deepwater Horizon disaster remains one of the largest oil spills in history. This event caused significant damage to coastal ecosystems, the full extent of which has yet to be fully determined. Crude oil contains toxic heavy metals and substances such as polycyclic aromatic hydrocarbons that are detrimental to some microbial species and may be used as food and energy resources by others. As a result, oil spills have the potential to cause significant shifts in microbial communities. This study assessed the impact of oil contamination on the function of endophytic microbial communities associated with saltmarsh cordgrass (Spartina alterniflora). Soil samples were collected from two locations in coastal Louisiana, USA: one severely affected by the Deepwater Horizon oil spill and one relatively unaffected location. Spartina alterniflora seedlings were grown in both soil samples in greenhouses, and GeoChip 5.0 was used to evaluate the endophytic microbial metatranscriptome shifts in response to host plant oil exposure. Oil exposure was associated with significant shifts in microbial gene expression in functional categories related to carbon cycling, virulence, metal homeostasis, organic remediation, and phosphorus utilization. Notably, significant increases in expression were observed in genes related to metal detoxification with the exception of chromium, and both significant increases and decreases in expression were observed in functional gene subcategories related to hydrocarbon metabolism. These findings show that host oil exposure elicits multiple changes in gene expression from their endophytic microbial communities, producing effects that may potentially impact host plant fitness.

Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolution than ever before using light microscopy. Here we describe the steps implemented to achieve the visualization of individual messenger RNAs (mRNA) using single-molecule FISH (smFISH) probes, as well as detection of mRNA/protein (mRNP) complexes via smFISH in combination with IF.