Oral Presentation 40th Annual Lorne Genome Conference 2019

Viral Noncoding RNAs: Diversity in Form and Function (#1)

Joan Steitz 1
  1. Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, CT, USA

Herpesviruses produce noncoding (nc)RNAs, some of which are essential to the viral life cycle.

One such ncRNA from Kaposi’s sarcoma-associated herpesvirus (KSHV) is the highly abundant polyadenylated nuclear (PAN) RNA, which is required for production and release of new viruses from infected cells. Although lacking nucleotide sequence conservation, PAN RNAs from the related viruses KSHV and RRV – when knocked down – exhibit the same phenotype, loss of late lytic viral gene expression and progeny virion production. We show that KSHV and RRV PAN RNAs can functionally substitute for each other to rescue this phenotype, but in contrast to published literature, the reduction in viral gene expression upon PAN RNA knockdown is not due to loss of PAN RNA association with conserved, specific chromatin loci. Instead, our data indicate that mRNA export from the nucleus is dependent on PAN RNA serving as a binding platform to sequester cellular proteins that are mislocalized to the nucleoplasm during infection.

Studies of another herpesviral ncRNA called HSUR1 from Herpesvirus saimiri (HVS) has provided insights into an important but poorly-understood cellular regulatory mechanism called target-directed miRNA decay (TDMD). Whereas argonaute (Ago) proteins use miRNAs as guides to identify and repress mRNAs, thereby shaping post-transcriptional gene expression, protective association with Agos can result in exceptionally long miRNA half-lives. Our mutational analysis of HSUR1 - a TDMD target of host miR-27 - has suggested that formation of a bipartite duplex is necessary for robust miRNA decay. The duplex is composed of both 5' (seed) and 3' paired miRNA regions connected by an unpaired flexible linker.  Strong complementarity to the miR-27 3' end results in tailing leading to production of stable miRNA isoforms (isomiRs), which are associated with all four human Agos. In collaboration with Ian MacRae’s lab at Scripps Institute, we have obtained X-ray structures of human Ago2 bound simultaneously to miRNAs and TDMD-inducing targets. They confirm the formation of bipartite duplexes.  Since Ago2 is physically too small to accommodate the 3'-paired duplex, the 3’ end of the miRNA is pulled out of its normal binding pocket in the PAZ domain.  It juts out into the solvent and Ago2 is trapped in a hyper-extended conformation. The Ago-released miRNA 3' end is then subjected to intracellular tailing and trimming, which we also observe when 3'-end binding by Ago is mutationally compromised.  These results will facilitate identification of additional viral and cellular TDMD targets, as well as the enzymes responsible for decay, leading to better understanding of how cellular miRNA populations can be regulated.