Functional characterization of viral non-polyadenylated sequences that enable mRNA stability and translation

Baylor College Of Medicine, Houston TX

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Eukaryotic mRNAs typically have a 5’ cap and 3’ poly(A) tail that protect the ends from degradation. In addition, the cap and poly(A) tail are recognized by specific RNA binding proteins (eIF4E and poly(A) binding protein [PABP], respectively) that help the mRNA form a closed loop that promotes efficient translation. Many viruses analogously ensure their mRNAs end in poly(A) tails, but there are thousands of other viruses, including Flaviviridae, Bunyavirales, Reovirales, and Nodaviridae, that solely generate non-polyadenylated mRNAs. A handful of these non-poly(A) mRNAs are known to bind PABP, while others instead use viral proteins to bridge the non-poly(A) end to proteins bound to the cap, enabling the mRNA to adopt a closed loop. However, key molecular details of how the vast majority of viral non-poly(A) 3’ ends, including from Bunyavirales, enable mRNA stability and translation remain unknown. Addressing this gap will reveal critical insights into molecular mechanisms of pathogenesis and identify novel avenues for developing highly specific antivirals. Bunyavirales is a large order of single-stranded, segmented negative-sense RNA viruses that can cause serious human disease, yet there are no effective therapies or vaccines. They generate capped, non-poly(A) mRNAs and the exact 3’ ends of some of these transcripts were mapped decades ago. Their regulation is independent of PABP, but little else is known about how exactly these 3’ ends are stabilized or enable translation. This is in part due to a lack of efficient methods for generating and manipulating non- poly(A) mRNAs in cells. We have now overcome this major obstacle by repurposing a unique cellular process discovered in our lab (3’ end processing of the noncoding RNA MALAT1) into a novel expression method that efficiently and easily generates non-poly(A) mRNAs that precisely end in any sequence of interest. Here, we will take advantage of this new method to characterize the key minimal sequences and underlying molecular mechanisms of how select viral non-poly(A) mRNAs are stabilized and translated. We hypothesize that many of these non-poly(A) sequences fold into structures that enable interactions with RNA binding proteins distinct from PABP, thereby enabling the viral mRNAs to be subjected to unique regulatory strategies that may promote their own expression over host mRNAs and/or be targetable by therapeutics. In Aim 1, we will define in detail how non-poly(A) 3’ ends from exemplar bunyaviral mRNAs enable mRNA functionality. The minimal functional sequences and their protein binding partners will be identified, thereby revealing new paradigms of bunyaviral post-transcriptional gene regulation. In Aim 2, we will perform a novel massively parallel reporter assay to screen non-poly(A) sequences from the 3’ ends of a variety of viral mRNAs in order to rapidly identify those that most efficiently stabilize a reporter mRNA and enable robust translation. These collective efforts will reveal critical insights into how viruses ensure their mRNAs are hyper-stable and translated, which will help guide the development of novel antivirals and mRNA-based therapeutics with higher expression and efficacy.