Periodic Reporting for period 3 - T-FRAME (Real-time analysis of ribosomal frameshifting and its impact on immunity and disease)
Periodo di rendicontazione: 2024-02-01 al 2024-09-30
RNA viruses like influenza and coronavirus spread around the world every year with the risk of pandemics. Other RNA viruses like HIV is no longer fatal for the developed world, yet is a chronic disease with no real cure. A striking feature of these viruses is that their mRNAs contain specific signals that direct a portion of translating ribosomes to move into an alternative reading frame. Programmed ribosome frameshifting is a well-conserved translational recoding event and is critical for the virulence and pathogenicity of RNA viruses. In addition to cis-acting RNA elements, it is suggested that there are numerous cellular factors and small RNAs involved in the regulation frameshifting events. However, how these interactions work during translation elongation and whether they can affect infection processes remains elusive.
In T-FRAME, we study regulation of recoding events during translation in the context of chronic viral infections and in respiratory viruses. Akin to other post transcriptional gene expression events, it is expected that there are regulatory molecules in the cell apart from the cis-acting RNA elements that determine levels of frameshifting in a time and tissue-specific manner. However, there is currently a lack of knowledge on the cellular regulators of this exquisite RNA-based gene regulatory event. To overcome this knowledge gap, T-FRAME aims to identify novel factors and study their interplay with viral RNA elements to elucidate molecular details of alternative translation strategies across different model systems including HIV-1 and SARS-CoV-2. Caliskan group is employing a highly interdisciplinary toolset ranging from single-molecule analysis to infection models.
T-FRAME will advance our understanding of how RNA structure and trans-factors shape translation regimes in higher eukaryotes and how deviations from the standard decoding path impact infection and innate immunity. I envision harnessing these findings to develop novel tools for synthetic biology and new design principles for RNA-centric antiviral and immune therapies. A comprehensive analysis of frameshift regulation will allow controlling and precisely targeting these RNA molecules, which I hope will eventually provide new design principles for RNA-centric antiviral and immune therapies.
In T-FRAME, we study regulation of recoding events during translation in the context of chronic viral infections and in respiratory viruses. Akin to other post transcriptional gene expression events, it is expected that there are regulatory molecules in the cell apart from the cis-acting RNA elements that determine levels of frameshifting in a time and tissue-specific manner. However, there is currently a lack of knowledge on the cellular regulators of this exquisite RNA-based gene regulatory event. To overcome this knowledge gap, T-FRAME aims to identify novel factors and study their interplay with viral RNA elements to elucidate molecular details of alternative translation strategies across different model systems including HIV-1 and SARS-CoV-2. Caliskan group is employing a highly interdisciplinary toolset ranging from single-molecule analysis to infection models.
T-FRAME will advance our understanding of how RNA structure and trans-factors shape translation regimes in higher eukaryotes and how deviations from the standard decoding path impact infection and innate immunity. I envision harnessing these findings to develop novel tools for synthetic biology and new design principles for RNA-centric antiviral and immune therapies. A comprehensive analysis of frameshift regulation will allow controlling and precisely targeting these RNA molecules, which I hope will eventually provide new design principles for RNA-centric antiviral and immune therapies.
It is well established that RNA molecules in the cell interact with a myriad of factors. Our overarching hypothesis is that viral frameshifting RNAs can be also regulated both directly and indirectly by cellular factors such as RNA binding proteins or non-coding RNAs during an infection. Our research questions focus on what regulatory proteins or other factors interact with frameshifting complexes, how they control translation pathways during viral infections and how these processes can be directly modulated. Ultimately, we aim to use this knowledge for designing antivirals targeting frameshifting RNAs during translation of viral RNAs. In the first funding period, we have established a frameshift interactome capture assay and discovered host encoded RNA-binding factors that interact with the frameshift RNAs in HIV-1 and SARS-CoV-2. Through these efforts, we identified several host-encoded candidate proteins that interact with the frameshift element. In one of the studies, we identified a human interferon-induced protein called the short isoform of the zinc-finger antiviral protein ZAP-S, as the direct regulator of frameshifting on SARS-CoV-2. ZAP-S is an RNA binding protein that specifically interact with the frameshift stimulating RNA pseudoknot of the SARS coronaviruses. During translation of the viral RNA, the interaction of ZAP-S with the frameshift RNA alters the stability of the secondary structure, which cannot fold into the pseudoknot essential for frameshifting. Ultimately, at high levels of ZAP-S, frameshift rates decrease, which lead to a drop in the viral polymerase level and consequently impede viral replication (Zimmer and Kibe et al., Nat. Comm., 2021).
Our mechanistic studies of the RNA interactions relies on using a combination of structural, biochemical and single molecule analysis tools. Using this toolset we have dissected interaction principles of frameshift RNA elements in unprecedented detail. We also integrated infection assays to study the functions of the trans-acting factors and the interplay between frameshift RNAs and the host and viral factors. Our interdisciplinary approach represents a benchmark to study the emerging concept of protein-mediated frameshifting events, which can open doors for new immune modulatory and antiviral intervention strategies. Highlights of our work are summarized below.
In the upcoming period, we will further develop our assays to directly visualize how cis and trans-acting elements interact with the translation apparatus in real-time.
Our mechanistic studies of the RNA interactions relies on using a combination of structural, biochemical and single molecule analysis tools. Using this toolset we have dissected interaction principles of frameshift RNA elements in unprecedented detail. We also integrated infection assays to study the functions of the trans-acting factors and the interplay between frameshift RNAs and the host and viral factors. Our interdisciplinary approach represents a benchmark to study the emerging concept of protein-mediated frameshifting events, which can open doors for new immune modulatory and antiviral intervention strategies. Highlights of our work are summarized below.
In the upcoming period, we will further develop our assays to directly visualize how cis and trans-acting elements interact with the translation apparatus in real-time.
The T-FRAME project initially focused on investigating frameshifting regulation and mechanisms in HIV-1 infected T-cells. However, with the emergence of the unexpected pandemic, our research expanded to include coronaviruses. Coronaviruses exhibit exceptionally high levels of frameshifting (25-40%), which produces the RNA-dependent RNA Polymerase of the virus crucial for their replication.
Our current research endeavors aim to uncover how trans-activators or repressors play a role in re-programming the reading frame in both HIV-1 and coronaviruses. In answering these questions, we went beyond the state of the art with our technical tools, established the first Crispr-i screen in HIV-1 and SARS-CoV-2 frameshifting, developed novel tools to analyse single molecule data and we are currently pushing technical limits to monitor translation in real time using novel single molecule analysis tools.
Discovery of new trans-acting factors changing the translational reading frame offers new avenues to inhibit viral replication and modulate host responses. From a practical standpoint, the results of Work Package 2 (WP2) will aid in the development of small peptides designed to target RNA structures, which could act as starting points for antiviral interventions.
Our current research endeavors aim to uncover how trans-activators or repressors play a role in re-programming the reading frame in both HIV-1 and coronaviruses. In answering these questions, we went beyond the state of the art with our technical tools, established the first Crispr-i screen in HIV-1 and SARS-CoV-2 frameshifting, developed novel tools to analyse single molecule data and we are currently pushing technical limits to monitor translation in real time using novel single molecule analysis tools.
Discovery of new trans-acting factors changing the translational reading frame offers new avenues to inhibit viral replication and modulate host responses. From a practical standpoint, the results of Work Package 2 (WP2) will aid in the development of small peptides designed to target RNA structures, which could act as starting points for antiviral interventions.