Elucidating the RNA-binding mechanisms and functions of E3 ubiquitin ligase TRIM25 in cell biology and innate immunity

Pathogenic viruses have a huge impact on human health and have caused numerous major epidemics both in the past and during the 21st century. The innate immune system is the body’s first line of defence against viruses, with pattern recognition receptors recognising molecules unique to viruses and triggering the expression of interferons and other anti-viral cytokines, leading to the formation of an anti-viral state. The Tripartite Motif Containing 25 (TRIM25) is an E3 ubiquitin ligase thought to be a key component in the activation of signalling by the pattern recognition receptor Retinoic Acid-Inducible Gene I Protein (RIG-I), which recognises viral RNAs with a 5’-triphosphate moiety. TRIM25 has recently been identified as an RNA-binding protein, raising the question of whether its RNA-binding activity is important for its role in innate immunity. In this thesis, I demonstrated that TRIM25’s RNA-binding activity is mediated by its C-terminal PRY/SPRY domain and is required for its E3 ligase activity. I also generated TRIM25 knockout cells using a CRISPR/Cas9 strategy in HeLa and HEK293 cell lines and showed that deletion of TRIM25 does not generally affect levels of the mRNA binding partners of TRIM25 identified by a genome-wide Cross-Linking Immunoprecipitation (CLIP) screen. Finally, I showed that although deletion of TRIM25 in HEK293 cells reduced their ability to restrict Influenza A virus infection, it did not affect activation of RIG-I signalling pathway in response to 5’- triphosphate RNA. This suggests that TRIM25 is redundant for RIG-I signalling in HEK293 cells and its role in restricting Influenza A virus infection is unrelated to its role in the RIG-I pathway. These findings have opened new lines of investigations into functional and molecular roles of TRIM25 in cell biology and control of pathogenic infections and I have generated tools to aid in these investigations

Lin28a uses distinct mechanisms of binding to RNA and affects positively and negatively miRNA levels

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3′-5′ exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing

The Grizzly, October 6, 2011

Fong, CPPC Developing Strategic Plan • Study Abroad Undergoes Major Changes, Relocation • Art Department Sponsors Trip to New York City • Residence Life Sponsors First-Ever Sexual Olympics • Senior Spends Summer Researching Parkinson\u27s • International Film Festival: A Glimpse Into Other Cultures • Wismer on Wheels Offers Helping Hand • Club Promotes Suicide Awareness • Anti-anxiety Group Aims to Relieve Stress • UC Club Soccer Kicks Off First Full Season • UC Fencing Club Looking to Increase its Numbers • Coach Profile: Mark Feinberg, Swimminghttps://digitalcommons.ursinus.edu/grizzlynews/1842/thumbnail.jp

The Grizzly, September 15, 2011

New CAB Events Coming This Fall • UC Commemorates 9/11 • CAB Hosts Activities Fair in Zack\u27s • GSA Welcomes New Board • Tips to Assist with Time Management • New Math Professor Brings Biostatistics Expertise • Peer Docent Program Returns to Berman • Senior Reminisces on Semester Spent Abroad • Opinions: Updated Sexual Assault Policy Needs Awareness; Libya Should Look to Spain When Rebuilding Government • Fast Start has Volleyball on Track for the Playoffs • Cross Country Eyes Continued Improvement in 2011 • Men\u27s Basketball Team Goes Abroadhttps://digitalcommons.ursinus.edu/grizzlynews/1839/thumbnail.jp

System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection

International audienceGraphical Abstract Highlights d A quarter of the RBPome changes upon SINV infection d Alterations in RBP activity are largely explained by changes in RNA availability d Altered RBPs are crucial for viral infection efficacy d GEMIN5 binds to the 5 0 end of SINV RNAs and regulates viral gene expressio

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Background TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Results Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Conclusions Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century