Interrogation of RNA-protein interaction dynamics in bacterial growth.

Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them are orthologs of human mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. For the first time, the dynamics of the bacterial RBPome have been interrogated, showcasing how this approach can reveal the function of uncharacterised proteins and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies.

System-wide analysis of RNA and protein subcellular localization dynamics.

Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.

Transgene codon usage drives viral fitness and therapeutic efficacy in oncolytic adenoviruses.

Arming oncolytic adenoviruses with therapeutic transgenes is a well-established strategy for multimodal tumour attack. However, this strategy sometimes leads to unexpected attenuated viral replication and a loss of oncolytic effects, preventing these viruses from reaching the clinic. Previous work has shown that altering codon usage in viral genes can hamper viral fitness. Here, we have analysed how transgene codon usage impacts viral replication and oncolytic activity. We observe that, although transgenes with optimized codons show high expression levels at the first round of infection, they impair viral fitness and are therefore not expressed in a sustained manner. Conversely, transgenes encoded by suboptimal codons do not compromise viral replication and are thus stably expressed over time, allowing a greater oncolytic activity both in vitro and in vivo. Altogether, our work shows that fine-tuning codon usage leads to a concerted optimization of transgene expression and viral replication paving the way for the rational design of more efficacious oncolytic therapies.

Codon Usage and Adenovirus Fitness: Implications for Vaccine Development.

Vaccination is the most effective method to date to prevent viral diseases. It intends to mimic a naturally occurring infection while avoiding the disease, exposing our bodies to viral antigens to trigger an immune response that will protect us from future infections. Among different strategies for vaccine development, recombinant vaccines are one of the most efficient ones. Recombinant vaccines use safe viral vectors as vehicles and incorporate a transgenic antigen of the pathogen against which we intend to generate an immune response. These vaccines can be based on replication-deficient viruses or replication-competent viruses. While the most effective strategy involves replication-competent viruses, they must be attenuated to prevent any health hazard while guaranteeing a strong humoral and cellular immune response. Several attenuation strategies for adenoviral-based vaccine development have been contemplated over time. In this paper, we will review them and discuss novel approaches based on the principle that protein synthesis from individual genes can be modulated by codon usage bias manipulation. We will summarize vaccine approaches that consider recoding of viral proteins to produce adenoviral attenuation and recoding of the transgene antigens for both viral attenuation and efficient viral epitope expression.

Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS).

RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.

Organic phase separation opens up new opportunities to interrogate the RNA-binding proteome.

Protein-RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic-aqueous phase separation-based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein-RNA interactions.

Author Correction: Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.

Translational reprogramming in tumour cells can generate oncoselectivity in viral therapies.

Systemic treatment of cancer requires tumour-selective therapies that eliminate cancer cells yet preserve healthy tissues from undesired damage. Tumoral transformation is associated with profound effects in translational reprogramming of gene expression, such that tumour-specific translational regulation presents an attractive possibility for generating oncoselective therapies. We recently discovered that mRNA translational control by cytoplasmic polyadenylation element-binding proteins (CPEBs) is reactivated in cancer. Here we present a novel approach to restrict genetic-engineered therapies to malignant tissues based on CPEB translational regulation of target mRNAs. We demonstrate that tumour reprogramming of CPEB-mediated mRNA stability and translational regulation modulates tumour-specific expression of viral proteins. For oncolytic adenoviruses, insertion of CPE regulatory sequences in the 3'-untranslated region of the E1A gene provides oncoselectivity, with full potency in cancer cells but attenuated in normal tissues. Our results demonstrate the potential of this strategy to improve oncolytic virus design and provide a framework for exploiting CPE-regulated transgenes for therapy.

Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

Genome-wide miR-155 and miR-802 target gene identification in the hippocampus of Ts65Dn Down syndrome mouse model by miRNA sponges.

BACKGROUND: Down syndrome (DS) or trisomy 21 is the result of a genetic dosage imbalance that translates in a broad clinical spectrum. A major challenge in the study of DS is the identification of functional genetic elements with wide impact on phenotypic alterations. Recently, miRNAs have been recognized as major contributors to several disease conditions by acting as post-transcriptional regulators of a plethora of genes. Five chromosome 21 (HSA21) miRNAs have been found overexpressed in DS individuals and could function as key elements in the pathophysiology. Interestingly, in the trisomic Ts65Dn DS mouse model two of these miRNAs (miR-155 and miR-802) are also triplicated and overexpressed in brain. RESULTS: In the current work, we interrogated the impact of miR-155 and miR-802 upregulation on the transcriptome of Ts65Dn brains. We developed a lentiviral miRNA-sponge strategy (Lv-miR155-802T) to identify in vivo relevant miR-155 and miR-802 target mRNAs. Hippocampal injections of lentiviral sponges in Ts65Dn mice normalized the expression of miR-155 and miR-802 and rescued the levels of their targets methyl-CpG-binding protein 2 gene (Mecp2), SH2 (Src homology 2)-containing inositol phosphatase-1 (Ship1) and Forkhead box protein M1 (FoxM1). Transcriptomic data of Lv-miR155-802T miRNA-sponge treated hippocampi correlated with candidate targets highlighting miRNA dosage-sensitive genes. Significant associations were found in a subset of genes (Rufy2, Nova1, Nav1, Thoc1 and Sumo3) that could be experimentally validated. CONCLUSIONS: The lentiviral miRNA-sponge strategy demonstrated the genome-wide regulatory effects of miR-155 and miR-802. Furthermore, the analysis combining predicted candidates and experimental transcriptomic data proved to retrieve genes with potential significance in DS-hippocampal phenotype bridging with DS other neurological-associated diseases such as Alzheimer's disease.

Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells.

Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3'UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3'UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses.