2024 Rocky Mountain RNA Symposium

Speaker Abstracts

In Order of Presentation



Homa Ghalei, PhD
Associate Professor
Department of Biochemistry
Emory University School of Medicine

 

Molecular Consequences of Deregulated rRNA Processing and Modification in Biology and Disease

 


 


Conner Purdy
Graduate Student, Ford Lab
University of Colorado-Anschutz 

Determining the Role of eIF3d/e-Mediated Stress-Induced Translation in Breast Cancer Metastasis

Authors: Stephen Connor Purdy, Kate Matlin, Chris Alderman, Rui Zhao, Neel Mukherjee, Heide Ford

Nearly all breast cancer-related deaths are caused by metastatic disease instead of the primary tumor. Unfortunately, there are currently no effective targeted therapies for metastasis. It has been shown that genomic alterations are not the main drivers of metastasis, and that instead, tumor cell plasticity, due to changes in gene expression in response to external cues, plays a critical role. While much research has focused on transcriptional/epigenetic means to induce tumor cell plasticity and metastasis, the role of protein translation in metastasis has been underappreciated. Importantly, recent studies have highlighted drastic differences in translational demand between tumor cells at the primary versus metastatic sites. The goal of this project is to understand the role of stress-induced translation in breast cancer metastasis, with a focus on eIF3e. eIF3e is one of 13 subunits of the eIF3 complex (eIF3a-m) involved in translation in mammalian cells. eIF3e, and its binding partner eIF3d, are critical for the translation of mRNAs that regulate key processes such as response to stress, epithelial to mesenchymal transition (EMT), proliferation and survival. These processes also play key roles in tumorigenesis and metastasis; and it is known that eIF3e is dysregulated in numerous cancers, including breast cancer. Excitingly, we recently showed that a novel compound, NCGC00378430 (abbreviated 8430), inhibits breast cancer-associated EMT and metastasis in vivo, and discovered that it binds to eIF3e. We show that 8430 does not alter global translation but can inhibit non-canonical translation of mRNAs such as c-Jun and HIF1⍺ during nutrient deprivation or hypoxia, respectively, similar to eIF3e, or its binding partner, eIF3d. We show that 8430 binds to eIF3e and decreases the amount of eIF3d within the eIF3 complex, without altering the composition of other subunits. Because 8430 targets eIF3e and inhibits EMT and metastasis, these data implicate eIF3d/e as important contributors to metastasis, and potential therapeutic targets. We have also developed closely related analogs of 8430 such as “209” which displays similar phenotypes in vitro and has better solubility and potency than 8430. To further understand the role of eIF3d/e in hypoxia and metastasis, we have recently performed ribosome sequencing (RiboSeq) experiments +/- hypoxia, +/- 209, and +/- eIF3e and eIF3d knockdown. This work implicates eIF3d/e in the regulation of specialized translation in response to stressful and changing microenvironments, such as hypoxia, during the metastatic cascade. Furthermore, our novel compounds, 8430 or 209 (and future analogs), may provide a starting point for determining whether inhibition of eIF3d/e will decrease tumor cell plasticity and metastasis, while conferring limited side effects.

 


 

Kristin Patrick, PhD
Assistant Professor
Microbial Pathogenesis and Immunology
Texas A&M College of Medicine

Nuclear RNA binding proteins: Concertmasters of the macrophage innate immune response

 


 

Kristin Fluke, PhD
Postdoctoral Researcher, Santangelo Lab
Colorado State University

Unique and extensive epitranscriptomic profiles in heat-loving Archaea enhance thermophily

Authors: Kristin A Fluke(1,3), Nan Dai(2), Yeuh-Lin Tsai(2), Ryan T Fuchs(2), Shing P Ho(3), Victoria Talbott(1), Hallie P Febvre(3), Liam Elkins(1), Eric J Wolf(2), Jackson Shiltz(3), Brett G Robb(2), Ivan R Correa Jr.(2), Thomas J Santangelo(3)

(1) Colorado State University, Cell and Molecular Biology, Fort Collins, CO, (2) New England Biolabs, RNA Division, Beverly, MA, (3) Colorado State University, Department of Biochemistry and Molecular Biology, Fort Collins, CO

The extraordinary quantity of known RNA modifications and their ubiquity in all life strongly suggests the epitranscriptome provides tangible benefits to cellular fitness. Ribosomal RNA is among the most heavily modified RNAs in a cell; modifications to rRNA are known to have profound impacts on ribosome function, and in turn, proteostasis. Recent investigations into archaeal epitranscriptomes have demonstrated that ribosomes from Thermococcus kodakarensis, a heat-loving archaeon, are densely modified with 4-acetylcytidine (ac4C) and 5-methylcytidine (m5C), and that the epitranscriptome supports hyperthermophilic growth. Using LC-MS/MS, bisulfite-sequencing, and high-resolution cryo-EM structures of the archaeal ribosome, we identified a unique epitranscriptomic mark in the T. kodakarensis 16S rRNA that includes a new RNA modification, m4,2C. We characterized and structurally resolved a novel class of RNA methyltransferase that generates m4,2C whose function is critical for hyperthermophilic growth. The phylogenetic distribution of the newly identified m4,2C synthase family implies m4,2C is biologically relevant in each Domain. Resistance of m4,2C to bisulfite-driven deamination suggests that efforts to capture m5C profiles via bisulfite sequencing are also capturing m4,2C.

 


 

Heather Hundley, PhD
Sagalowsky Professor of Biology
Associate Professor of Biology
Indiana University

Mechanisms of in vivo Target Recognition by the ADAR family of RNA Modification Enzymes

 


 

Laura White, PhD
Research Associate, Hesselberth Lab
University of Colorado-Anschutz

So many mods in so little time: >45 RNA modifications profiled by direct RNA-seq

Authors: Laura White, Kezia Dobson, Jill Bilodeaux, Samantha Del Pozo, Saylor Strugar, Shelby Andersen, Amber Baldwin, Chloe Barrington, Nadine Kortel, Federico Martinez Seidel, Kristin Watt, Neel Mukherjee, and Jay Hesselberth

Epitranscriptomic marks on nucleic acids produce disruptions in ion flow when they are fed through biological nanopores. In principle, this effect enables the identification of any modification that generates a differentiable signal distortion; however, distinguishing the signals produced by the >170 distinct chemical modifications present on RNA molecules is a non-trivial technical challenge. We leveraged the diverse chemical repertoire of tRNAs, the most abundantly modified class of RNA, to evaluate the signals produced at known modification sites across a broad range of viral, prokaryotic, and eukaryotic species. We evaluated signals from more than 45 distinct RNA modifications using both first and second generation Oxford Nanopore direct RNA sequencing chemistry, and further report a proof of concept approach for detecting low abundance mitochondrial and viral tRNA reads using the higher library throughputs enabled by the new RNA004 chemistry. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across the tree of life using nanopore sequencing.

 


 

Eliza Lee, PhD
Postdoctoral Associate, Cech Lab
University of Colorado Boulder

N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5’ Splice Site Motifs

Authors: Eliza S. Lee (1,5), Harrison. W. Smith (1), Sean S. J. Ihn (1), Leticia Scalize de Olivera (1), Yifang E. Wang (1), Robert Y. Jomphe (1,2), Syed Nabeel-Shah (3,4), Shuye Pu (4), Jack F. Greenblatt (3,4), Alexander F. Palazzo (1)

(1) University of Toronto, Department of Biochemistry, Canada; (2) Cell Biology Program, Hospital for Sick Children, Toronto, Canada; (3) University of Toronto, Department of Molecular Genetics, Canada; (4) Terrence Donnelly Centre for Cellular & Biomolecular Research, Toronto, Canada; (5) Present Address: Department of Biochemistry, University of Colorado Boulder, CO

In eukaryotes, quality control of mRNA represents an important regulatory mechanism for gene expression. Misprocessed mRNAs that contain an intact 5’ Splice Site (5’SS) motif are retained in the nucleus and targeted for decay. Previously, we showed that the nuclear retention of these transcripts requires ZFC3H1, a component of the Poly(A) Exosome Targeting (PAXT) complex, and U1-70K, a component of the U1 snRNP. In S. pombe, the ZFC3H1 homolog, Red1, binds to the YTH-domain containing protein Mmi1 to target certain RNA transcripts for nuclear retention and decay. Here we show that ZFC3H1 and U1-70K interact with YTHDC1 and YTHDC2, two YTH domain-containing proteins that bind to N-6-methyladenosine (m6A) modified RNAs. We then show that YTHDC1 and YTHDC2 are required for the nuclear retention of mRNAs with intact 5’SS motifs. Furthermore, disruption of m6A methyltransferase activity inhibits the nuclear retention of these transcripts. Using m6A-miCLIP analysis, we map m6A methylation marks to intronic polyadenylated (IPA) transcripts, which contain intact 5’SS motifs and are nuclear retained and degraded in a ZFC3H1-dependent manner. We find that m6A is enriched near intact 5’SS motifs and the poly(A)-tail. Overall, this work suggests that the m6A modification acts as part of an evolutionarily conserved quality control mechanism that targets misprocessed mRNAs for nuclear retention and decay.

 


 

Myriam Gorospe, PhD
Chief, Laboratory of Genetics and Genomics
National Institute on Aging Intramural Research Program, NIH

Long noncoding RNA LANCL1-AS1 in aging muscle regeneration

 


 

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