The dietary specialist fruit fly Drosophila sechellia has evolved to specialize on the toxic fruit of its host plant Morinda citrifolia. Toxicity of Morinda fruit is primarily due to high levels of octanoic acid (OA). Using RNA interference (RNAi), prior work found that knockdown of Osiris family genes Osiris 6 (Osi6), Osi7, and Osi8 led to increased susceptibility to OA in adult D. melanogaster flies, likely representing genes underlying a Quantitative Trait Locus (QTL) for OA resistance in D. sechellia. While genes in this major effect locus are beginning to be revealed, prior work has shown at least five regions of the genome contribute to OA resistance. Here, we identify new candidate OA resistance genes by performing differential gene expression analysis using RNA-sequencing (RNA-seq) on control and OA-exposed D. sechellia flies. We found 104 significantly differentially expressed genes with annotated orthologs in D. melanogaster, including six Osiris gene family members, consistent with previous functional studies and gene expression analyses. Gene ontology (GO) term enrichment showed significant enrichment for cuticle development in upregulated genes and significant enrichment of immune and defense responses in downregulated genes, suggesting important aspects of the physiology of D. sechellia that may play a role in OA resistance. In addition, we identified five candidate OA resistance genes that potentially underlie QTL peaks outside of the major effect region, representing promising new candidate genes for future functional studies.

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codonsthe ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN) n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

MPW) ¶ These authors contributed equally to this work. AbstractLevels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons--the ramp region--of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN) n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with largescale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

I would first like to thank Dr. Michael Weir, who has positively influenced the lives of countless students through his roles as a teacher, mentor and faculty advisor. I remember being told by a random senior to take Intro Bio with Dr. Weir my freshman year. I couldn't be gladder that I did. It was his class that encouraged me to change my mind from majoring in chemistry and switching to bio. I recall being led to consider things like a scientist for the first time. Over the years, he has guided me through designing and executing experiments and has done it all with an attention to detail and understanding of the big picture simultaneously. I genuinely don't think I could have asked for a more patient, supportive, and enthusiastic research mentor.Past and present Weir lab members have also been extremely helpful. Previous work from Jacob Glickman led to the questions that this project addresses. Previous lab members Samuel Roth, Brynne Lycette, and Tae Hee Kim and current lab members Desmond Yao and Elliot Williams have offered thoughtful insight during lab meetings. I would like to thank Claire Fournier, Miin Lin, and Mary Vallo for teaching me proper wet-lab protocol and more when I first joined the lab. I would also like to thank Will Barr, Om Chatterji, and Felix Cram with planning and carrying out experiments and for being wonderful lab mates. I would also like to send the biggest thank you to Karen Voelkel-Meiman who honestly deserves her own page given the amount of times I have waltzed up to her with no warning and she just had the answers, as she always does. I will miss her wit and wisdom immensely.

The idea that base pairing between mRNAs and structural rRNAs of ribosomes might contribute to protein translation has long been an intriguing possibility. The 530 loop of 16S rRNA has been implicated in translation initiation, elongation and termination. This loop and the corresponding highly‐conserved loop in 18S rRNA are located in the mRNA entrance tunnel of ribosomes. As noted before high‐resolution ribosome structures were described, the 530 loop contains a 3‐nucleotide‐repeating pattern complementary to the 3‐nucleotide periodicity of protein open reading frames characterized by overrepresentation of (GCN)n. We find that the 3‐nucleotide periodicity is significantly enhanced downstream of translation start codons of highly expressed Saccharomyces cerevisiae genes with significant depression of G at nucleotides 2 and 3 of these codons. We propose that during translocation steps, exposed rRNA nucleotides of the 530 loop can transiently base pair with the second and third mRNA nucleotides of the incoming A‐site codon before engagement of the A‐site tRNA and that this promotes efficient launching of the ribosome through the start region codons permitting high protein expression. This cooperation between the A‐site and 530 loop requires a precise positioning of the mRNA reading frame, which if compromised in several adjacent codons leads to low translation levels.Support or Funding InformationNational Institutes of Health 1R15GM096228Beckman Scholars Program, Arnold & Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.