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
Ribosome biogenesis is a complex and highly regulated process involving the action of over 200 assembly factors bringing together a total of 79 proteins and 4 ribosomal RNAs in yeast. The maturation of rRNAs from precursor transcripts is a critical aspect of ribosome biogenesis involving a series of orchestrated processing steps by nucleases and over a hundred RNA chemical modifications. Despite tight regulation and quality controls, rRNA processing and chemical modifications go awry in several human diseases. Missense mutations in genes encoding structural subunits of the RNA exosome, the ribonuclease complex required for rRNA processing, cause a growing family of diseases with diverse pathologies, collectively termed RNA exosomopathies. Similarly, pathogenic mutations that impact rRNA modifications are associated with neurodevelopmental disorders and implicated in the onset and progression of cancer. I will present data on recent findings made by my research team and our collaborators that shed light on the molecular consequences of dysregulated rRNA processing and modifications. These molecular defects have a profound impact on both the quantity and quality of ribosomes within the translating pool, thereby disrupting the delicate balance of cellular protein homeostasis. Our findings underscore the intricate interplay between ribosome biogenesis, RNA processing, modifications, and disease pathogenesis, providing crucial insights and a deeper understanding of these complex cellular processes.
Connor Purdy
Doctoral 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
When innate immune cells like macrophages encounter a pathogen, they massively upregulate hundreds of inflammatory and antimicrobial genes in a matter of minutes. This response is executed by specialized transcription factors (e.g. NFkB, IRF3, AP-1) that are activated by pathogen sensing signaling cascades. Under this current “transcription-focused” paradigm, the potential contribution of post-transcriptional control mechanisms to tuning inflammatory gene expression has been largely neglected. My lab has implicated several RNA binding proteins (RBPs) in the SR and hnRNP families of splicing factors in controlling the kinetics and amplitude of inflammatory gene expression. Collectively, our work highlights RNA processing as a key regulatory node in shaping the macrophage response to pathogens.Recently, we have started investigating ways that macrophages could quickly remodel their nuclei to redistribute RBPs and prioritize processing of inflammatory genes. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. We found that the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, is a critical component of the macrophage antimicrobial response. Specifically, we report that lipopolysaccharide (LPS) treatment triggers dynamic remodeling of macrophage paraspeckles and that loss of paraspeckles, via Neat1 KO, results in damped pro-inflammatory responses and a failure to control replication of both bacterial and viral pathogens. We are currently working to understand how paraspeckles are remodeled in response to additional pathogen-associated molecular patterns and how other nuclear MLOs respond to pathogen-mediated cues.
Collectively, our work supports a model whereby dynamic assembly and disassembly of MLOs reorganize the nuclear landscape to enable macrophage responses to different pathogens.
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
The ADAR family of RNA binding proteins binds double-stranded RNA (dsRNA) and catalyzes the deamination of adenosine (A) to inosine (I). As the A-to-I conversion changes hydrogen bonding specificity of the base, most enzymes and cellular factors recognize inosine as guanosine. Hence, A-to-I RNA editing can alter the coding potential, splicing, and small RNA-mediated silencing of mRNA. The ability of ADARs to change the genome-encoded information present in RNA provides an important means to diversify the transcripts expressed in an organism’s tissues over time and is being harnessed for personalized medicine approaches to correct mutations at the RNA level and improve human health. However, details of how ADARs bind specific transcripts as well as how certain adenosines are selected within the bound transcript for editing remain largely unknown, especially at a molecular level in vivo. Using the C. elegans model system, we have been able to demonstrate that the A-to-I editing machinery involves RNA binding specificity dictated by one protein, while the enzymatic activity is contributed by a second protein. Our insights gained from recent enhanced crosslinking and immunoprecipitation coupled to high-throughput sequencing specific experiments will be presented. In addition, recent work on how editing and RNA binding of transcripts in specific tissues is influenced by the environment and development will be discussed.
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
With advancing age, the skeletal muscle experiences a gradual loss of function resulting from declining number, quality, and size of muscle fibers. In turn, the loss of muscle is associated with frailty, fractures, and increased risk of metabolic diseases. Muscle mass and function are maintained through myogenesis, a process whereby muscle stem cells regenerate muscle fibers in the adult. As skeletal muscle ages, declining function of muscle stem cells (satellite cells) to form myofibers contributes to the loss of muscle mass (sarcopenia). Given that muscle function and regeneration are increasingly compromised with age, we have a long-standing interest in understanding myogenesis.Among the many regulators of myogenesis, our lab has focused on RNA-binding proteins (RBPs) and noncoding (nc)RNAs for many years. In this presentation, I discuss some of the long noncoding RNAs [1] implicated in controlling myogenesis that we have studied over the past 5 years. Human myogenesis was found to be fostered by lncRNA OIP5-AS1, abundant in the cytoplasm of myoblasts, which scaffolds HuR and MEF2C mRNA, thereby enhancing the production of myogenic transcription factor MEF2C [2]. The same lncRNA, OIP-AS1, was further discovered to lower miR-7 levels by target-directed microRNA degradation (TDMD); the loss of miR-7 derepressed production of the fusogenic protein MYMX, which is crucial for myoblast fusion and myotube generation [3]. During human myogenesis, a linear lncRNA residing in the nucleus, lncFAM, was found to accumulate and promote differentiation by recruiting HNRNPL to the MYBPC2 promoter, in turn rising MYBPC2 levels [4]. Interestingly, we also found a circular lncRNA (circSamd4) that promoted myogenesis by increasing during myogenesis, as circSamd4 inactivated the transcriptional repressors PURA and PURB and enabled transcription of the Myh gene [5].
In an ongoing study, we have focused on lncRNAs directly implicated in skeletal muscle aging. We examined the transcriptomes of skeletal muscle biopsies from a cross-sectional study of healthy individuals 22 to 83 years old to identify lncRNAs changing in expression levels. After contrasting them with the transcriptomes changing during human myogenesis in culture, a top candidate emerged, LANCL1-AS1, which increased with myogenesis and declined with muscle aging. Interestingly, silencing LANCL1-AS1 attenuated myogenesis and globally reduced the levels of mRNAs transcribed from the mitochondrial genome (mt-mRNAs). Molecular characterization revealed that LANCL1-AS1 associated with the mitochondrial RBP LRPPRC, which, together with SLIRP, regulates mt-mRNA levels by preserving the length of their poly(A) tails and prolonging their half-lives. Accordingly, overexpressing LANCL1-AS1, but not a mutant LANCL1-AS1 unable to interact with LRPPRC, restored myogenic capacity to primary myoblasts from muscle of old monkeys, where LANCL1-AS1 is conserved. In light of the finding that mt-mRNAs in old human skeletal muscle had shorter poly(A) tails than those in young muscle, we propose that LANCL1-AS1 helps to preserve myogenesis across the life span by improving mitochondrial function.
[1] Herman et al., Mol Cell (2022). https://pubmed.ncbi.nlm.nih.gov/35714586/
[2] Yang et al., Nucleic Acids Res (2020). https://pubmed.ncbi.nlm.nih.gov/33270893/
[3] Yang et al., Nucleic Acids Res (2022). https://pubmed.ncbi.nlm.nih.gov/35736212/
[4] Chang et al., Nucleic Acids Res (2023). https://pubmed.ncbi.nlm.nih.gov/36533518/
[5] Pandey et al., Nucleic Acids Res (2020). https://pubmed.ncbi.nlm.nih.gov/31980816/