In Order of Presentation
Sigrid Nachtergaele, PhD
Gene expression regulation by RNA modifications
Chemical modification of RNA is a critical mechanism of gene expression regulation, controlling RNA processing, stability, and, in the case of mRNA, translation. The chemical diversity of RNA modifications suggests that there is extensive biology yet to be uncovered. A recent surge in this field has been driven by rapid advances in high throughput sequencing methods that allow us to map these marks on a transcriptome-wide scale. However, many of these studies remain correlative and are not able to reveal the molecular mechanisms of RNA modification function or regulation. The information we glean from such studies often represents an average across all transcripts in the cell, and does not take into account the spatial organization or temporal control of RNA transcription, processing, and trafficking. Moreover, the majority of work on mRNA modifications has focused on a single modification, N6-methyladenosine (m6A), which represents only one of over one hundred modifications annotated to date. Our work seeks to address fundamental questions about mRNA modifications, to expand beyond m6A-based studies, and to develop the tools to study a broader diversity of RNA modifications and their functions.
Dylan Parker, PhD
University of Colorado-Boulder
G3BP1 functions as an RNA condenser by forming RNA-RNA interactions in stress granules
RNP granules are a well-studied class of biomolecular condensates requiring both RNA and proteins for their assembly. However, the cellular mechanisms and molecular interactions driving separation of RNP granules from the bulk cytoplasm remains incompletely understood. Stress granules are RNP granules that form upon stress induced increases in non-translating mRNPs, and due to their reversibility are a tractable model for understanding RNP granule assembly. The G3BP proteins are critical factors in stress granule assembly. A variety of mechanisms have been postulated for stress granule organization focusing primarily on the multivalent interactions of G3BP1 and G3BP2 with other stress granule proteins and RNA to form mesoscale RNP granules. We now demonstrate an additional role for G3BP1 in triggering RNP granule assembly by promoting the formation of intermolecular RNA::RNA interactions. We show that after the initial formation of RNP granules, G3BP1 is dispensable both in vitro and in vivo for persistence of the RNA component of granules suggesting that G3BP1 is only required after initial stress granule formation to counteract RNA decondensing machinery such as the ATP-driven DEAD-box proteins.[DP1] These results identify a general mechanism by which RNP granule-specific RNA binding proteins promote RNP granule assembly through "RNA condenser" activity by catalyzing the formation of intermolecular RNA interactions. Moreover, the stability of RNA-only granules in the absence of RNA binding proteins highlights the need for active mechanisms to limit condensate stability and lifetime. This finding has significant implications for diseases related to excessive formation and runaway aggregation of RNP granules.
Doctoral Student, Osborne Nishimura Lab
Colorado State University
Drugging the embryo: How eggshell permeabilization helps us investigate the cytoskeletal impact on mRNA movement
Translation typically occurs in the cytoplasm or the endoplasmic reticulum. However, erm-1 (ezrin/radixin/moesin) mRNA concentrates at the plasma membrane where the protein it encodes connects the plasma membrane to the actin cytoskeleton. This molecular event serves to coordinate cell shape changes. Indeed, other transcripts that encode membrane-associated proteins also concentrate at the plasma membrane, but neither the mechanisms directing their localization nor the reasons for their local translation are understood. Previously, we determined that erm-1 mRNA localization to the membrane is translation-dependent and directed by its encoded N-terminal FERM-domain. Here, we test whether erm-1/ERM-1 localization occurs through active or passive mechanisms, and we explore which cytoskeletal components are required for their transport. Therefore, we developed an eggshell permeabilization strategy, followed by drug treatment and then gentle smFISH. Using this approach, we tested the effect of Nocodazole (microtubule loss) or Cytochalasin D (actin loss) on mRNA localization. Using C. elegans, we can further investigate whether motor proteins are involved in mRNA transport process using both smFISH and live mRNA imaging techniques. Our data suggests that dynein, but not kinesin, perturbs RNA localization, though still unclear whether this effect is direct or indirect. This work is relevant because impaired mRNA localization in neurons and other cell types causes disease but studying mRNA localization in disease-specific models is challenging. Furthermore, the process of directing translation-dependent localization of mRNA to plasma membranes is a distinct and novel method of performing local translation, one whose mechanisms and impacts we aim to better understand.
Doctoral Student, Rissland Lab
University of Colorado-Anschutz
Potent regulation of gene expression by the open reading frame
Non-optimal synonymous codon usage hinders gene expression, but the mechanisms by which this occurs are poorly understood. We and others have previously shown that non-optimal codons slow translation elongation speeds and thereby trigger mRNA degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting there are additional mechanisms by which codon usage regulates gene expression. Using reporter assays in human and Drosophila cells, we found that transcript levels account for less than half of the variation in protein abundance. This discrepancy is explained by translational differences. With bulk and single-molecule imaging assays, we show that non-optimal transcripts are bound by fewer ribosomes, and reduced translation initiation is responsible. Non-optimal transcripts are also less bound by the key translation initiation factors eIF4E and eIF4G, providing a mechanistic explanation for their reduced initiation rates. Importantly, repression can occur in the absence of mRNA decay and doesn’t depend on the non-optimality sensor, CNOT3. Our results reveal a potent new mechanism of regulation by codon usage, where non-optimal codons repress further rounds of translation.
Post-transcriptional gene regulation in vertebrates
The Bazzini-lab studies how genes are regulated at the post-transcriptional level in vertebrates. Basically, what molecular mechanism dictate the stability and the level of translation of mRNA in human cells and zebrafish embryos? And how these regulations affect development (zebrafish embryos) and human diseases? Specifically, we are looking for brilliant students interesting in working in translation regulation mediated by small ORF. Our group demonstrated that translation affect mRNA stability in a codon dependent manner in zebrafish embryos and human cells, a mechanism called codon optimality. Then, we discovered that translation of small ORF in the 3’UTR (dORF) enhances translation of the canonical main ORF in zebrafish and human cells. And recently, we found the first method to trigger mRNA knockdowns in zebrafish embryos based on a Cas13d system. These are the three main research avenues of the lab.
Derrick Morton, PhD
University of Southern California
Investigating RNA dysregulation in complex brain disorders
The complexity of the transcriptome poses a challenge to cells because they must manage RNA processing events - degrading surplus and defective RNAs - without destroying functional RNA molecules. Remarkably, a multi-subunit post-transcriptional regulatory machine, the RNA exosome, performs these two divergent RNA processing roles: functional maturation and quality-control driven destruction. The RNA exosome complex is an essential and ubiquitous ribonuclease that is critical for proper processing and degradation of a variety of cellular RNAs. How the RNA exosome mediates precise processing of some RNA targets, yet complete destruction of others, is not understood. A currently favored theory is that distinct RNA exosome co-factors recruit specific RNAs to the complex. However, it is not known whether the same cofactors also guide RNA exosome activity on recruited RNAs. The recent discovery that recessive mutations in genes encoding distinct structural RNA exosome subunits cause a variety of neurodevelopmental disorders makes defining the role of the RNA exosome in neurodevelopment critically important to understand the basis of these diseases. The primary research goal of my lab is to study the fundamental mechanisms of post-transcriptional regulation of gene expression with an emphasis on RNA processing factors mutated in complex brain disorders. Specifically, we are interested in the post-transcriptional activities of the RNA exosome in human neurodevelopment and disease. Thus, we have taken the strategy of coupling in vivo Drosophila genetics with in vitro human iPSC-derived brain organoid models to understand how defects in subunits of the ubiquitous RNA exosome complex cause tissue-specific consequences.
MD/PhD Student, Fantauzzo Lab
University of Colorado-Anschutz
Alternative RNA splicing downstream of PDGFRα signaling in craniofacial development
Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and over a thousand differential alternative RNA splicing events. Here, we demonstrate via enhanced crosslinking and immunoprecipitation (eCLIP)-seq analysis of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons. Further, an unbiased motif enrichment analysis of Srsf3 binding sites revealed a loss of binding to canonical Srsf3 CA-rich motifs in stimulated samples, which could be due to changes in ribonucleoprotein composition or loss of RNA-binding due to electrostatic repulsion. Through analysis of complementary RNA-seq data, we show that the subset of transcripts that are bound by Srsf3 and undergo alternative splicing upon PDGFRα signaling commonly encode regulators of Wnt signaling, a pathway known to be critical for mammalian craniofacial development. Taken together, these findings provide considerable insight into the mechanisms underlying gene expression regulation during craniofacial development.
Postdoctoral Fellow, Kieft Lab
University of Colorado-Anschutz
Structural basis for translation termination-reinitiation at overlapping open reading frames in viruses
The paradigm for eukaryotic translation dictates that each mRNA contains a single open reading frame (ORF) encoding one protein, but growing evidence suggests translation frequently occurs outside the main coding region. Under certain circumstances, failed ribosome recycling events lead to translation of a downstream ORF through reinitiation. Certain viruses, including norovirus and influenza B virus, use RNA structures embedded in the upstream coding region to induce reinitiation events and express essential viral proteins. These reinitiation-promoting viral RNA structures contain sequences that allow direct interactions with the ribosome following termination. The structure of reinitiation-promoting RNA representatives from different viruses as well as their mechanism of action have remained largely uncharacterized. By performing homology searches we found this RNA motif in viruses beyond those previously described. The highly-conserved base paired portions and the location of the ribosome binding nucleotides of multiple representatives of this viral RNA motif are supported by chemical probing data. Translation assays in lysate using dual luciferase reporters reveal that RNAs from different viruses reinitiate with variable efficiency due to differences in the RNA structure, relative affinity for the 40S subunit, and the stop codon context. Using single-particle cryoEM analysis, we determined the structure of a reinitiation-promoting viral RNA in complex with a mammalian 80S ribosome, confirming the binding site of the RNA on the 40S subunit. This structure lays the groundwork to understand how this class of viral RNA can prevent complete ribosome recycling and promote a reinitiation event at a specific downstream start codon.
Susan (Sichen) Shao, PhD
Mechanisms of small molecule translation inhibitors
Translation termination is an essential cellular process that is also of therapeutic interest for diseases that manifest from nonsense mutations. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes, and facilitates ribosome recycling. Several small molecules have been reported to trigger eRF1 degradation, which in turns improves the translational readthrough of premature stop codons. However, the mechanism of action of these small molecules is poorly understood. Here, we report cryogenic electron microscopy (cryo-EM) structures showing that one such molecule glues the N domain of eRF1 involved in stop codon recognition to the ribosomal subunit interface near the decoding center. Aberrant stabilization of eRF1 on ribosomes leads to eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings raise new considerations for pharmacologically targeting translation termination.