Standing your ground to exoribonucleases: Function of Flavivirus long non-coding RNAs

Abstract

Members of the Flaviviridae (e.g., Dengue virus, West Nile virus, and Hepatitis C virus) contain a positive-sense RNA genome that encodes a large polyprotein. It is now also clear most if not all of these viruses also produce an abundant subgenomic long non-coding RNA. These non-coding RNAs, which are called subgenomic flavivirus RNAs (sfRNAs) or Xrn1-resistant RNAs (xrRNAs), are stable decay intermediates generated from the viral genomic RNA through the stalling of the cellular exoribonuclease Xrn1 at highly structured regions. Several functions of these flavivirus long non-coding RNAs have been revealed in recent years. The generation of these sfRNAs/xrRNAs from viral transcripts results in the repression of Xrn1 and the dysregulation of cellular mRNA stability. The abundant sfRNAs also serve directly as a decoy for important cellular protein regulators of the interferon and RNA interference antiviral pathways. Thus the generation of long non-coding RNAs from flaviviruses, hepaciviruses and pestiviruses likely disrupts aspects of innate immunity and may directly contribute to viral replication, cytopathology and pathogenesis.

Keywords: Exoribonuclease; Flavivirus; IRES; Long non-coding RNA; RNA decay; RNA stability; Untranslated regions.

Fig. 1

An overview of the general pathways of cytoplasmic mRNA decay in eukaryotic cells. The degradation of mRNAs is generally initiated by deadenylation via the CCR4-NOT deadenylase complex (as well as some contribution from other deadenylases). As seen in the center portion of the diagram, deadenylated mRNAs then generally undergo decapping by DCP2 to generate a 5′ monophosphate containing RNA. This decapped mRNA is then rapidly and processively degraded by the 5′-3′ exonuclease Xrn1. Xrn1-mediated mRNA decay can feedback in some fashion to the nucleus where it helps to buffer mRNA synthesis at the level of transcription to maintain cellular homeostasis. As seen on the right side of the diagram, deadenylated mRNAs can also be degraded by an alternative 3′-5′ exonucleolytic pathway mediated by the Dis3 enzyme associated with the RNA exosome or an independent exonuclease called Dis3L2. Finally (left side of the diagram), if an mRNA is subjected to endonucleolytic cleavage (e.g., by RNAi-mediated decay, RNAse L digestion, etc.), the newly formed and fully accessible 5′ monophosphate and 3' OH containing RNA fragments are rapidly degraded by the two exonucleolytic pathways as indicated.

Fig. 2

LncRNAs formed by Xrn1 stalling on flavivirus transcripts. Panel A. Xrn1-mediated exonucleolytic decay of the positive sense genomic RNA of arthropod-borne flaviviruses (e.g., Dengue virus or West Nile virus) generates a set of sfRNAs from the 3′ UTR. The 5′ end of each of the depicted RNA products represent Xrn1 stalling just 5′ to knot-like tertiary structures formed by higher order folding of the depicted stem loops (Roby et al., 2014). Panel B. Xrn1-mediated exonucleolytic decay of the positive sense genomic RNA of HCV generates a set of sfRNAs from the 5′ UTR. Reconstitution assays using recombinant Xrn1 generate the three RNA products depicted. The 5′ end of each of the RNA products represent Xrn1 stalling just 5′ to the indicated secondary structure landmarks (Moon et al., 2015).

Fig. 3

Xrn1 stalling at flaviviral RNA structures represses enzymatic activity in a reversible fashion. Left panel: cellular mRNAs are degraded rapidly in a processive fashion by Xrn1. The enzyme is naturally released when it is finished degrading the RNA and can rapidly begin degrading other RNA substrates. Right panel. Xrn1 stalls at knot-like RNA structures while degrading flavivirus transcripts and is only slowly released from the RNA substrate. This results in reversible inhibition of Xrn1 while the enzyme remains associated with the flaviviral RNA decay intermediate. Once Xrn1 is released, the enzyme can go onto degrade other RNAs and the long non-coding flavivirus RNA can perform its function(s) (e.g., as a decoy for cellular RNA binding proteins in the interferon pathway).