Periodic Reporting for period 1 - T-FRAME (Real-time analysis of ribosomal frameshifting and its impact on immunity and disease)
Reporting period: 2021-02-01 to 2022-07-31
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. Here, the main challenges are:
- Identifying putative host cellular and viral factors that modulate translation of frameshift RNAs.
- Defining structural features of cis- and trans-acting frameshift regulators, and elucidating how they impede translation elongation.
- characterizing how frameshift regulators impact infections and immune response.
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.
The main objectives of the T-FRAME project are:
Understanding the mechanism of protein-mediated frameshifting in eukaryotes
Discovery of novel host and viral factors that regulate translation of frameshifting genes
Defining the scope and dynamics of frameshifting in immune cells upon infection or activation by interferons.
- Identifying putative host cellular and viral factors that modulate translation of frameshift RNAs.
- Defining structural features of cis- and trans-acting frameshift regulators, and elucidating how they impede translation elongation.
- characterizing how frameshift regulators impact infections and immune response.
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.
The main objectives of the T-FRAME project are:
Understanding the mechanism of protein-mediated frameshifting in eukaryotes
Discovery of novel host and viral factors that regulate translation of frameshifting genes
Defining the scope and dynamics of frameshifting in immune cells upon infection or activation by interferons.
Efficient frameshifting is mediated by stimulatory RNA structures and slippery sequences embedded in the mRNA. In addition, viral or host encoded proteins and small molecules can be involved in the regulation of frameshifting. To uncover the molecular mechanism and principles of frameshifting, we initially focused on two recently discovered viral and host factors, 2A protein from cardioviruses and SHFL protein, which is a host interferon induced gene product that impair HIV-1 frameshift. We hypothesized that the interactions of the factors can be direct, occurring by virtue of altering the stability and dynamics of the frameshift stimulatory RNA structures. Such an interaction can help to create a suitable time window for codon:anticodon base pair interactions to be reestablished in the new reading frame during translation elongation. To test this hypothesis, we initially focused our efforts to understand the structural and mechanistic features of the cardiovirus 2A proteins and their interplay with frameshift RNAs from two cardiovirus species, Encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelitis virus (TMEV). Despite sharing functional similarity, structurally the two proteins were found to be highly divergent. We have shown that the EMCV 2A protein and TMEV 2A protein both interact with the respective RNA targets with high affinity and specificity. Mutating only one residue within the loop region of the stimulatory hairpin (CCC-> CUC) would impair the binding of 2A protein as well as frameshift stimulation (Hill et al. 2021a, Hill et al. 2021b). On the other hand binding of SHFL to the HIV-1 RNA and other frameshift RNAs were unspecific. SHFL has also shown no specificity to other frameshift RNA structures (unpublished). We suppose that SHFL acts on stalled ribosomal complexes and not on frameshifting complexes, per se. Due to this non-specificity, we decided to focus initially on solving the mechanistic features of direct frameshift modulators, like the cardiovirus 2A protein.
Our next aim was to mechanistically characterize the interactions of cardiovurus 2A and EMCV RNA in more detail. Interactions between RNA and interacting partners are usually short-lived, or weak making it difficult to decipher the underlying physical principles and precise control mechanisms. In order to gain an in-depth insight into RNA dynamics, we employ state of the art optical tweezers with high-resolution imaging and microfluidics. The combination of these techniques allows us to obtain spatial and dynamic information on RNA structures, which lead to translation pauses and the movement of ribosomes into the –1 reading frame.
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.
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Our next aim was to mechanistically characterize the interactions of cardiovurus 2A and EMCV RNA in more detail. Interactions between RNA and interacting partners are usually short-lived, or weak making it difficult to decipher the underlying physical principles and precise control mechanisms. In order to gain an in-depth insight into RNA dynamics, we employ state of the art optical tweezers with high-resolution imaging and microfluidics. The combination of these techniques allows us to obtain spatial and dynamic information on RNA structures, which lead to translation pauses and the movement of ribosomes into the –1 reading frame.
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.
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