Universal method to co-profile RNA synthesis in host and pathogen

PI(s)/Head responsible for the resource:

Anniina Vihervaara

Host organisation(s):

KTH Royal Institute of Technology

Resource description:

Pathogen genomes can be DNA or RNA, single- or double-stranded, segmented, or contiguous, linear, or circular, and their mRNA encoded by plus or minus strand. The fundamental molecular diversity within pathogen species challenges development of universal tools and treatments, urgently needed for research, medicine, and pandemic preparedness. Here, we build on our state-of-the-art expertise in nascent RNA sequencing, method development, and cellular responses to stress, including viral and bacterial infections. We develop a universally applicable method to track RNA synthesis simultaneously in pathogen and host using Precision Run-On (PRO) chemistry. In PRO-seq, a single biotin-coupled ribonucleotide is incorporated into the 3’-ends of growing RNA chains, labelling the precise positions of engaged RNA Polymerases (Pols) genome-wide. My laboratory develops PRO techniques, and tracks transcription by eukaryote Pols I-III, mitochondrial Pol, and bacterial Pol (Fig. 1A-E). Recently, we obtained a proof-of-principle for monitoring RNA synthesis by RNA templated RNA Pol (RdRP) in Sindbis virus (Fig. 1E), showing the universality of our approach across species and genomes. Importantly, we detect on-going RNA synthesis simultaneously in virus and its host, enabling dynamic analyses of transcription, RNA virus replication, and host responses. Here, we sediment our preliminary work into host-pathogen PRO-seq (hpPRO-seq) method, using human cells infected with RNA viruses. With the Rothfuchs laboratory, we extent hpPRO-seq to various infectious pathogens, including SARS-CoV-2 (ssRNA+), HIV-1 (retro), and monkeypox (dsDNA) viruses, and mycobacterium tuberculosis. hpPRO-seq reveals dynamics of host-pathogen interactions, tracks instant and sustained responses in the host, screens species-specificities of drugs, sequences pathogen genomes, traces lineages and mutations, and is compatible with patient-derived material - infected with existing or emerging pathogens.

Contact information:

Anniina Vihervaara
Department of Gene Technology, KTH Royal Institute of Technology
Email: anniina.vihervaara@scilifelab.se