RNA levers and switches controlling viral gene expression.

RNA viruses are diverse and abundant pathogens that are responsible for numerous human diseases. RNA viruses possess relatively compact genomes and have therefore evolved multiple mechanisms to maximize their coding capacities, often by encoding overlapping reading frames. These reading frames are then decoded by mechanisms such as alternative splicing and ribosomal frameshifting to produce multiple distinct proteins. These solutions are enabled by the ability of the RNA genome to fold into 3D structures that can mimic cellular RNAs, hijack host proteins, and expose or occlude regulatory protein-binding motifs to ultimately control key process in the viral life cycle. We highlight recent findings focusing on less conventional mechanisms of gene expression and new discoveries on the role of RNA structures.

Web-based platform for analysis of RNA folding from high throughput chemical probing data.

RNA structures play critical roles in regulating gene expression across all domains of life and viruses. Chemical probing methods coupled with massively parallel sequencing have revolutionized the RNA structure field by enabling the assessment of many structures in their native, physiological context. Previously, we developed Dimethyl-Sulfate-based Mutational Profiling and Sequencing (DMS-MaPseq), which uses DMS to label the Watson-Crick face of open and accessible adenine and cytosine bases in the RNA. We used this approach to determine the genome-wide structures of HIV-1 and SARS-CoV-2 in infected cells, which permitted uncovering new biology and identifying therapeutic targets. Due to the simplicity and ease of the experimental procedure, DMS-MaPseq has been adopted by labs worldwide. However, bioinformatic analysis remains a substantial hurdle for labs that often lack the necessary infrastructure and computational expertise. Here we present a modern web-based interface that automates the analysis of chemical probing profiles from raw sequencing files (http://rnadreem.org). The availability of a simple web-based platform for DMS-MaPseq analysis will dramatically expand studies of RNA structure and aid in the design of structure-based therapeutics.

The Cas10 nuclease activity relieves host dormancy to facilitate spacer acquisition and retention during type III-A CRISPR immunity.

A hallmark of CRISPR immunity is the acquisition of short viral DNA sequences, known as spacers, that are transcribed into guide RNAs to recognize complementary sequences. The staphylococcal type III-A CRISPR-Cas system uses guide RNAs to locate viral transcripts and start a response that displays two mechanisms of immunity. When immunity is triggered by an early-expressed phage RNA, degradation of viral ssDNA can cure the host from infection. In contrast, when the RNA guide targets a late-expressed transcript, defense requires the activity of Csm6, a non-specific RNase. Here we show that Csm6 triggers a growth arrest of the host that provides immunity at the population level which hinders viral propagation to allow the replication of non-infected cells. We demonstrate that this mechanism leads to defense against not only the target phage but also other viruses present in the population that fail to replicate in the arrested cells. On the other hand, dormancy limits the acquisition and retention of spacers that trigger it. We found that the ssDNase activity of type III-A systems is required for the re-growth of a subset of the arrested cells, presumably through the degradation of the phage DNA, ending target transcription and inactivating the immune response. Altogether, our work reveals a built-in mechanism within type III-A CRISPR-Cas systems that allows the exit from dormancy needed for the subsistence of spacers that provide broad-spectrum immunity.

The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The mammalian mitochondrial genome encodes thirteen oxidative phosphorylation system proteins, crucial in aerobic energy transduction. These proteins are translated from 9 monocistronic and 2 bicistronic transcripts, whose native structures remain unexplored, leaving fundamental molecular determinants of mitochondrial gene expression unknown. To address this gap, we developed a mitoDMS-MaPseq approach and used DREEM clustering to resolve the native human mitochondrial mt-mRNA structurome. We gained insights into mt-mRNA biology and translation regulatory mechanisms, including a unique programmed ribosomal frameshifting for the ATP8/ATP6 transcript. Furthermore, absence of the mt-mRNA maintenance factor LRPPRC led to a mitochondrial transcriptome structured differently, with specific mRNA regions exhibiting increased or decreased structuredness. This highlights the role of LRPPRC in maintaining mRNA folding to promote mt-mRNA stabilization and efficient translation. In conclusion, our mt-mRNA folding maps reveal novel mitochondrial gene expression mechanisms, serving as a detailed reference and tool for studying them in different physiological and pathological contexts.