Analyses of zebrafish and Xenopus oocyte maturation reveal conserved and diverged features of translational regulation of maternal cyclin B1 mRNA

<p>Abstract</p> <p>Background</p> <p>Vertebrate development relies on the regulated translation of stored maternal mRNAs, but how these regulatory mechanisms may have evolved to control translational efficiency of individual mRNAs is poorly understood. We compared the translational regulation and polyadenylation of the cyclin B1 mRNA during zebrafish and <it>Xenopus </it>oocyte maturation. Polyadenylation and translational activation of cyclin B1 mRNA is well characterized during <it>Xenopus </it>oocyte maturation. Specifically, <it>Xenopus </it>cyclin B1 mRNA is polyadenylated and translationally activated during oocyte maturation by proteins that recognize the conserved AAUAAA hexanucleotide and U-rich Cytoplasmic Polyadenylation Elements (CPEs) within cyclin B1 mRNA's 3'<b>U</b>n<b>T</b>ranslated <b>R</b>egion (3'<b>UTR</b>).</p> <p>Results</p> <p>The zebrafish cyclin B1 mRNA was polyadenylated during zebrafish oocyte maturation. Furthermore, the zebrafish cyclin B1 mRNA's 3'UTR was sufficient to stimulate translation of a reporter mRNA during zebrafish oocyte maturation. This stimulation required both AAUAAA and U-rich CPE-like sequences. However, in contrast to AAUAAA, the positions and sequences of the functionally defined CPEs were poorly conserved between <it>Xenopus </it>and zebrafish cyclin B1 mRNA 3'UTRs. To determine whether these differences were relevant to translation efficiency, we analyzed the translational activity of reporter mRNAs containing either the zebrafish or <it>Xenopus </it>cyclin B1 mRNA 3'UTRs during both zebrafish and <it>Xenopus </it>oocyte maturation. The zebrafish cyclin B1 3'UTR was quantitatively less effective at stimulating polyadenylation and translation compared to the <it>Xenopus </it>cyclin B1 3'UTR during both zebrafish and <it>Xenopus </it>oocyte maturation.</p> <p>Conclusion</p> <p>Although the factors that regulate translation of maternal mRNAs are highly conserved, the target sequences and overall sequence architecture within the 3'UTR of the cyclin B1 mRNA have diverged to affect translational efficiency, perhaps to optimize levels of cyclin B1 protein required by these different species during their earliest embryonic cell divisions.</p

Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling

Creation and Characterization of LRB (Light-Response BTB) IPIF (Phytochrome-Interacting Factor) Mutant Lines in Arabidopsis thaliana

Color poster with text, graphs, charts, and images.In order to better understand how the LRB and PIF genes interact we are taking a genetic approach, creating plants with T (transfer)-DNA disruptions of both LRB and PIF genes. Study of the phenotypes of these plants may shed light on how these two families of genes work together to regulate light responses or plant growth and development in general.National Science Foundation-Research in Undergraduate Institutions (RUI Grants #1354438); Unversity of Wisconsin--Eau Claire Differential Tuition; University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Can These Mutations Lead to Blindness? : Analyzing the Effects of Genetic Variants of Uncertain Significance

Color poster with text, charts, and images.Genetic testing involves examining a patient's DNA sequence to look for changes (mutations) in the DNA that can potentially cause disease or illness. While some of the changes are benign, many have not yet been characterized and are thus classified as variants of uncertain significance (VUS). In collaboration with Prevention Genetics, our lab has begun to analyze VUS that are predicted to affect the splicing of genes related to ocular diseases. Disruption or alteration of splicing can affect gene function and lead to disease. We are using a minigene system in which the gene segment under investigation is cloned into a plasmid vector and then transfected into eukaryotic cell culture. The mRNA transcripts are then collected and analyzed to determine the effects of the variant on the transcript. Analysis of VUS could lead to a change in variants’ interpretation and directly have an impact on patients and their families by supporting diagnosis and access to treatment.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Humidity as a non-pharmaceutical intervention for influenza A.

Influenza is a global problem infecting 5-10% of adults and 20-30% of children annually. Non-pharmaceutical interventions (NPIs) are attractive approaches to complement vaccination in the prevention and reduction of influenza. Strong cyclical reduction of absolute humidity has been associated with influenza outbreaks in temperate climates. This study tested the hypothesis that raising absolute humidity above seasonal lows would impact influenza virus survival and transmission in a key source of influenza virus distribution, a community school. Air samples and objects handled by students (e.g. blocks and markers) were collected from preschool classrooms. All samples were processed and PCR used to determine the presence of influenza virus and its amount. Additionally samples were tested for their ability to infect cells in cultures. We observed a significant reduction (p < 0.05) in the total number of influenza A virus positive samples (air and fomite) and viral genome copies upon humidification as compared to control rooms. This suggests the future potential of artificial humidification as a possible strategy to control influenza outbreaks in temperate climates. There were 2.3 times as many ILI cases in the control rooms compared to the humidified rooms, and whether there is a causal relationship, and its direction between the number of cases and levels of influenza virus in the rooms is not known. Additional research is required, but this is the first prospective study suggesting that exogenous humidification could serve as a scalable NPI for influenza or other viral outbreaks