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Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3′ termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal. We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem–loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

A trigger for gene silencing is caused by RNAs pairing together via structural changes., Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Polycomb Repressive Complex 2 (PRC2) contributes to initiation of heterochromatin through catalysis of histone H3 lysine 27 (H3K27) methylation. Human PRC2 can work productively with specific long noncoding RNAs (lncRNAs) at certain target loci, yet the underlying mechanism is unclear. In apparent contradiction to lncRNA-mediated PRC2 activity, PRC2 binds to RNA in a low-specificity, high-affinity manner, effectively competing with chromatin and making RNA a potent catalytic inhibitor of H3K27 methylation activity. We address this contradiction by testing the model that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target genes can generate a context that is favorable for PRC2 activity. This RNA matchmaking activity is promoted by the heterogenous nuclear ribonucleoprotein (hnRNP) B1, which uses multiple protein domains to bind to specific regions of HOTAIR via multi-valent protein-RNA interactions. Using chemical probing, we find that RNA-RNA interaction and B1 binding facilitate RNA structural changes in HOTAIR. Finally, we demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity. By dissecting molecular details of RNA matchmaking within the HOTAIR mechanism, we highlight a switch that can occur as PRC2 samples RNA interactions broadly in the nucleus, being activated on chromatin by specific RNA matches. This concept may generalize to many contexts where RNA plays a role in potentiating PRC2 activity.

Programmed −1 ribosomal frameshifts (−1 PRFs) are commonly used by viruses to regulate their enzymatic and structural protein levels. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that uses two independent −1 PRFs to express viral enzymes critical to establishing new HTLV-1 infections. How the cis-acting RNA elements in this viral transcript function to induce frameshifting is unknown. The objective of this work was to conclusively define the 3′ boundary of and the RNA elements within the HTLV-1 pro-pol frameshift site. We hypothesized that the frameshift site structure was a pseudoknot and that its 3′ boundary would be defined by the pseudoknot's 3′ end. To test these hypotheses, the in vitro frameshift efficiencies of three HTLV-1 pro-pol frameshift sites with different 3′ boundaries were quantified. The results indicated that nucleotides included in the longest construct were essential to highly efficient frameshift stimulation. Interestingly, only this construct could form the putative frameshift site pseudoknot. Next, the secondary structure of this frameshift site was determined. The dominant structure was an H-type pseudoknot which, together with the slippery sequence, stimulated frameshifting to 19.4(±0.3)%. The pseudoknot's critical role in frameshift stimulation was directly revealed by examining the impact of structural changes on HTLV-1 pro-pol −1 PRF. As predicted, mutations that occluded pseudoknot formation drastically reduced the frameshift efficiency. These results are significant because they demonstrate that a pseudoknot is important to HTLV-1 pro-pol −1 PRF and define the frameshift site's 3′ boundary.

The Tudor domain of human PHF1 recognizes trimethylated lysine 36 of histone H3 (H3K36me3). This interaction modulates methyltransferase activity of the PRC2 complex and plays a role in retention of PHF1 at the DNA damage sites. We have previously determined the structural basis for the association of Tudor with a methylated histone peptide. Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP). Using a combination of TROSY NMR and FRET we show that Tudor concomitantly interacts with H3K36me3 and DNA. Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins. Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.