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    Characterization of fungal RTG2 genes in retrograde signaling of Saccharomyces cerevisiae
    (2013) Ünlü, Ercan Selçuk; Narayanan, Lakshmi; Gordon, Donna M.
    Changes in the functional status of mitochondria result in the transcriptional activation of a subset of nuclear-encoded genes in a process referred to as retrograde signaling. In Saccharomyces cerevisiae, this molecular link between mitochondria and the nuclear genome is controlled by three key signaling proteins: Rtg1p, Rtg2p, and Rtg3p. Although the retrograde signaling response has been well characterized in S. cerevisiae, very little is known about this pathway in other fungi. In this study, we selected four species having uncharacterized open reading frames (ORFs) with more than 66% amino acid identity to Rtg2p for further analysis. To determine whether these putative RTG2 ORFs encoded bona fide regulators of retrograde signaling, we tested their ability to complement the defects associated with the S. cerevisiae rtg2? mutant. Specifically, we tested for complementation of citrate synthase (CIT2) and aconitase (ACO1) at the transcript and protein levels, glutamate auxotrophy, and changes in the interaction between Rtg2p and the negative regulator Mks1p. Our findings show that all four Rtg2p homologs are functional upon activation of retrograde signaling, although their degree of complementation varied. In addition, all Rtg2p homologs showed a marked reduction in Mks1p binding, which may contribute to their altered responses to retrograde signaling. © 2013 The Authors.
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    Small RNA sequencing based identification of MiRNAs in daphnia magna
    (Public Library Science, 2015) Ünlü, Ercan Selçuk; Gordon, Donna M.; Telli, Murat
    Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs) which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.
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    Transcriptional response and functional target analyses of miRNA and mRNA to planktivorous fish kairomone exposure in Daphnia magna
    (E Schweızerbartsche Verlagsbuchhandlung, 2021) Telli, Murat; Gordon, Donna M.; Ünlü, Ercan Selçuk
    The discovery of regulatory functions for microRNAs (miRNA) and recently described miRNA profiles for Daphnia (Brachiopoda, Cladocera) make it an ideal system to probe for epigenetic regulation of predator avoidance mechanisms in aquatic ecosystems. In this study we report that exposing D. magna to kairomone released by predatory fish from birth to the first reproduction resulted in the differential expression of the four miRNAs tested: miR-7, miR-34, miR-317, and miR-375. Normalized transcript levels for each miRNA were found to vary across the exposure period with no clear conserved pattern of expression despite functional target analyses by Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicating that predicted miRNA target genes are likely involved in related biological activities. Analysis of mRNA transcripts (Hsp70, Hsp90 (HSP = heat shock protein), actin, AKT-interacting protein (AKT), Glycogen synthase (GYS) and Insulin growth factor receptor (IGFR)) identified in the literature as kairomone-mediated genes, were also carried out. Similar to that obtained for miRNAs, the mRNA relative expression levels showed varying degrees of temporal regulation across the exposure time with the two heat shock transcripts exhibiting elevated levels at early and late time points of exposure while the AKT, GYS, and IGFR transcripts had a general decrease in relative expression during the first 96 hours. Differential mRNA expression data supports the conclusion that kairomone mediated response is regulated by cost of general biological processes and that of survival under long-term kairomone stress. These studies lay the foundation for future work aimed at linking miRNAs and their target transcripts to changes in the signaling events that govern Daphnia response to kairomone specific stress.