RNA-protein interactions in human health and disease

RNA-binding proteins (RBPs) are key molecules that control gene expression signaling through RNAprotein interactions. Consequently, they contribute to cellular homeostasis, normal development, and most human diseases. Importantly, new RBPs are being discovered by high-throughput proteomics, but we still have a limited understanding of their function. A pioneering study identified 860 RBPs in HeLa cells (Castello et al., Cell, 2012). Remarkably, more than 300 of these proteins were not known for their RNA-binding properties and bear no identifiable RNA-binding domains. Several other studies identified hundreds of novel RBPs in various cells and tissues. Despite the extensive cataloging of novel RBPs, we have a limited understanding of what dictates their molecular functions and RNA-related roles in human health and disease

Functional and structural characteristics of novel RBPs and RNA-protein interactions in the innate immune response to RNA viruses

RNA viruses have caused several epidemics in the 21st century. SARS-CoV-2 virus, which causes COVID-19, has already taken the lives of 2.8 million people and erased trillions of USD from the global economy. Another well-known RNA virus, influenza A virus, kills up to 500,000 people annually and causes a significant global socioeconomic burden. These are only two examples of many RNA viruses that cause severe problems for human health and the economy. Thus, a detailed molecular understanding of host-virus interactions is imperative to learn how best to inactivate the virus and prevent major disruptions. Finally, viruses have been used to uncover some of the most important cellular processes, such as mRNA splicing, capping, polyadenylation, and RNA interference, and they continue to provide insights into molecular phenomena that can improve our understanding of the basic biology of living organisms. We recently discovered and started characterizing a novel RNA binding protein, the E3 ubiquitin ligase TRIM25 (Choudhury et al., Cell Rep, 2014; Choudhury et al., BMC Biology, 2017). TRIM25 belongs to a large family of tripartite motif-containing proteins (more than 80), most of which have E3 ubiquitin ligase activity. All TRIMs have in common an amino-terminal tripartite domain arrangement (RING–Bbox1/2–coiled coil), but they differ in their C-terminal domains, which categorize them into several subtypes. Many TIRIMs are positive or negative regulators of innate immune pathways (i.e., a first-line defense against such pathogens as viruses). Importantly, TRIM25 is emerging as a key factor in the innate immune response to RNA viruses (including influenza A virus, severe acute respiratory syndrome, Sindbis virus, and dengue virus, among many others). Despite the essential involvement of TRIM25 in viral RNA-induced innate immune pathways, its RNA-binding functions are still poorly understood.

The most widely reported TRIM25 function involves activation of the pattern-recognition receptor RIG-I, which senses 5’-triphosphate (5’-ppp) moieties on viral RNAs and activates the innate immune response. Upon binding to 5’-ppp-RNA, RIG-I undergoes TRIM25-mediated K63 ubiquitination. This activates a signaling cascade that culminates in the phosphorylation of interferon regulatory factor 3 (IRF-3), IRF-7, and nuclear factor-κB, which translocate to the nucleus and induce type I interferon expression. Importantly, the purported role of TRIM25 in the RIG-I pathway has been recently challenged by numerous reports. We discovered that TRIM25 is an RBP that regulates the stability of host RNA (Choudhury et al., Cell Rep, 2014). We also revealed that TRIM25 binds RNA through a novel RNA-binding domain that resides in the PRY/SPRY region and showed that RNA binding appears to be crucial for its E3 ubiquitin ligase activity (Choudhury et al., BMC Biol, 2017). Despite this, the direct binding of TRIM25 to viral RNAs, its 3D structure in complex with host or viral RNAs, and its detailed function in cell biology and innate immunity have not yet been described. Additionally, the RNA-binding potential of other TRIMs with PRY/ SPRY domains has not yet been explored.

Within the same theme, we recently co-authored a study that identified global changes in RBP composition and affinity in response to Sindbis virus infection (Garcia-Moreno et al., Mol Cell, 2019). Over 200 RBPs showed a differential association with RNA upon infection with Sindbis virus. This remodeling of RNA-protein interactions is driven by the host cell to limit virus infection and by the virus to limit the host’s innate immune response.

Our most recent work uncovered the RNA-binding roles of TRIM25 in the innate immune response to RNA virus infections. In our recent publication, we revealed a new mechanism whereby TRIM25 binds to influenza A mRNA and destabilizes it, contributing to the antiviral response (Choudhury et al., Nucleic Acids Res, 2022, Fig. 1). Concomitantly, we aim to solve the E3 ubiquitin ligase TRIM25/RNA complex structure, which will inform us about novel RNA-binding domains and the role of RNA in stimulating ubiquitination. The outcome of this research will reveal how TRIM25 uses its RNA-binding activity for anti-viral functions. Importantly, TRIM25 belongs to a large (> 80 member) family of tripartite motif containing proteins that have various functions in cellular processes and disease, including development, apoptosis, autophagy, carcinogenesis, and innate immunity. Thus, our research will open new lines of investigation into other TRIM proteins and their putative RNA-binding roles.

Figure 1. Model of TRIM25 sensing and inhibiting IAV infection by controlling viral mRNA stability. During IAV infection, TRIM25 binds to positive strand RNAs and triggers mRNA degradation, which in turn might be one of the factors that contributes to inhibition of viral replication. Our results also predict additional, E3 ligase-independent mechanisms of TRIM25-mediated control of IAV infection (Choudhury et al., Nucleic Acids Res, 2022 doi: 10.1093/nar/gkac512).

Regulation of microRNAs through RBPs for the treatment of Parkinson’s disease

MicroRNAs (miRs) are small noncoding RNAs that negatively regulate the expression of mRNAs. They have defined tissue expression patterns and affect many cellular processes and developmental pathways. Most miRs are transcribed by RNA polymerase II, with a long primary transcript, termed pri-miR, that carries a hairpin structure. The biogenesis of miRs is accomplished by two enzymes, DROSHA and DICER, that catalyze two processing events in the nucleus (from pri-miR to pre-miR) and cytoplasm (from pre-miR to miR duplex), respectively. miR duplexes are incorporated into the RNA-induced silencing complex (RISC) together with Argonaute (AGO) protein, where one strand is selected to become the mature miR. The RISC then recognizes a specific mRNA sequence by complementary base-pairing, resulting in translation inhibition and/or RNA degradation.

Because of the important role of miRNAs in the control of gene expression and organism development, the production of mature miRNAs is tightly regulated at multiple levels, including transcriptional and post-transcriptional steps. We have shown that the biogenesis of miRs can be regulated posttranscriptionally by RBPs that bind to their pri-miRs and pre-miRs (Michlewski et al., Mol Cell, 2008; Michlewski and Caceres, Nat Struct Mol Biol, 2010; Choudhury et al., Genes Dev, 2012; Nowak et al., Nat Commun, 2014; Nowak et al., RNA, 2017; Kooshapur et al., Nat Commun, 2018; Michlewski and Caceres, RNA, 2019; Downie Ruiz Velasco et al., Mol Ther Nucleic Acids, 2019; Sajini et al., Nat Commun, 2019). We are currently working on regulation of the miR-7 pathway, which is directly involved in Parkinson’s disease (PD). Parkinson’s disease is an incurable neurodegenerative disease that affects all ages but is most prevalent in the elderly, afflicting over 1% of the population over the age of 60. Approximately 10 million people live with PD, and hundreds of thousands die from the disease each year. One of the main causes of PD is the overproduction and aggregation of α-synuclein (α-Syn) protein in brain cells in affected individuals. A large body of evidence indicates that decreases in α-Syn levels should be beneficial for PD patients. Several clinical trials are now focusing on α-Syn clearance by silencing RNAs or vaccines. Notably, miR-7 has been shown to target α-Syn production, and approaches for miR-7 replacement therapies have been proposed.

We have shown that HuR (ELAVL1) protein is a naturally occurring inhibitor of miR-7 production that binds to its pri-miR-7 (Choudhury et al., Genes Dev, 2012). Furthermore, evidence suggests that HuR is upregulated in PD, and binding to the α-Syn mRNA 3’-untranslated region stabilizes the transcript, thereby allowing an increase in α-Syn production. This suggests that disrupting the RNA/HuR complex will have a positive effect on miR-7 and negative effect on α-Syn, suggesting a novel approach for PD therapy. Indeed, we recently reported that the natural compound quercetin inhibited RNA/HuR and downregulated α-Syn levels in human cultured cells (Zhu et al., Nucleic Acids Res, 2021, Fig. 2).

Figure 2. Inhibition of HuR-RNA interactions lowers down the levels of alpha-Syn protein. (A) HuR inhibits the biogenesis of alpha-Syn targeting miR-7. (B) HuR stabilises alpha-Syn mRNA resulting in high levels of alpha-Syn protein. (C) Small molecule quercetin blocks the interaction of HuR with pri-miR-7 increasing the production of miR-7. (D) Quercetin blocks the interaction of HuR with alpha-Syn mRNA lowering down the levels of alpha-Syn protein (Zhu et al., Nucleic Acids Res, 2021, doi: 10.1093/nar/gkab484.).

In recent years, we have been developing a novel drug discovery platform, based on RNA-protein interactions in eukaryotic cell extracts with confocal nanoscanning (RP-CONA), to screen for specific compounds that interfere with the pri-miR-7/ HuR complex, thus reactivating miR-7 biogenesis, suppressing α-Syn production, and alleviating PD symptoms (Zhu et al., Nucleic Acids Res, 2021). Our strategy is based on an ultra-sensitive RNA-protein interaction assay in cell extracts from human cultured cells (Choudhury and Michlewski et al., Methods, 2019) and detecting pri-miR-7/ HuR complex disruption by a confocal nanoscanner (Fig. 2). The use of human cultured cell extracts for an ultrasensitive on-bead assay offers great advantages over traditional systems for drug screening that use purified proteins from prokaryotic cells. First, all human proteins, such as HuR, are extensively modified in human cells, which directly influences their structure, activity, and function. Second, RNA-protein complexes, such as pri-miR-7/ HuR, are analyzed in the context of other competing pri-miR-7/protein complexes, better reflecting the cellular environment. Our new methodology will allow the discovery of compounds that disrupt the pri-miR-7/HuR complex as close to the human cellular context as possible. Finally, implementation of the RP-CONA screening platform will allow many researchers to explore the possibilities of targeting RNA-protein interactions in research and medical-based projects.