Dynamic bulge nucleotides in the KSHV PAN ENE triple helix provide a unique binding platform for small molecule ligands

Abstract

Cellular and virus-coded long non-coding (lnc) RNAs support multiple roles related to biological and pathological processes. Several lncRNAs sequester their 3′ termini to evade cellular degradation machinery, thereby supporting disease progression. An intramolecular triplex involving the lncRNA 3′ terminus, the element for nuclear expression (ENE), stabilizes RNA transcripts and promotes persistent function. Therefore, such ENE triplexes, as presented here in Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) lncRNA, represent targets for therapeutic development. Towards identifying novel ligands targeting the PAN ENE triplex, we screened a library of immobilized small molecules and identified several triplex-binding chemotypes, the tightest of which exhibits micromolar binding affinity. Combined biophysical, biochemical, and computational strategies localized ligand binding to a platform created near a dinucleotide bulge at the base of the triplex. Crystal structures of apo (3.3 Å) and ligand-soaked (2.5 Å) ENE triplexes, which include a stabilizing basal duplex, indicate significant local structural rearrangements within this dinucleotide bulge. MD simulations and a modified nucleoside analog interference technique corroborate the role of the bulge and the base of the triplex in ligand binding. Together with recently discovered small molecules that reduce nuclear MALAT1 lncRNA levels by engaging its ENE triplex, our data supports the potential of targeting RNA triplexes with small molecules.

Crystal structure of a highly conserved enteroviral 5′ cloverleaf RNA replication element

“…Within the four-way junction, with no unpaired nucleotides and perfect cross-strand stacking of the P1 helix G10 with P4 helix G47 and P2 helix C11 with P3 helix C46, each set of these coaxial stacking forms almost a continuous A-form helix. As expected, the L2 loop closes the P2 helix and binds the Fab BL3-6, consistent with the Fab-loop interactions observed for other RNA-BL3-6 complex crystal structures 46 , 47 , 49 , 50 , 52 , 53 . Similarly, a trinucleotide loop L3 and a tetranucleotide loop L4 close the P3 and P4 helices, respectively.…”

“…Among others, Fab BL3-6 has been a very effective chaperone to crystallize and determine the high-resolution crystal structures of several RNA targets 46 , 47 , 49 , 50 , 52 , 53 . The Fab binds to a hairpin RNA with 5′-AAACA-3′ pentaloop sequence closed preferably by a G-C pair, which allows facile engineering of the epitope sequence into the target RNA 46 , 47 , 49 , 50 , 52 , 53 .…”

“…Our efforts to crystallize this WT 5′CL construct were unsuccessful; therefore, we followed a chaperoneassisted RNA crystallization strategy. We have been developing and employing Fab fragments as chaperones for Fab-RNA complex crystallization and as molecular replacement models for initial phasing during the structure determination process [46][47][48][49][50][51] . One of the approaches in this strategy utilizes a Fab-epitope module, which involves grafting the RNA epitope into the target RNA to enable its complex formation with the Fab, allowing subsequent crystallization of the Fab-RNA complex [46][47][48][49][50][51] .…”

“…We have been developing and employing Fab fragments as chaperones for Fab-RNA complex crystallization and as molecular replacement models for initial phasing during the structure determination process [46][47][48][49][50][51] . One of the approaches in this strategy utilizes a Fab-epitope module, which involves grafting the RNA epitope into the target RNA to enable its complex formation with the Fab, allowing subsequent crystallization of the Fab-RNA complex [46][47][48][49][50][51] . Among others, Fab BL3-6 has been a very effective chaperone to crystallize and determine the high-resolution crystal structures of several RNA targets 46,47,49,50,52,53 .…”

LncRNA NEAT1 Potentiates SREBP2 Activity to Promote Inflammatory Macrophage Activation and Limit Hantaan Virus Propagation

“…We found that the expression of NEAT1, especially NEAT1-2, was obviously induced from 12 to 36 hpi in both primary human and mouse monocytes and macrophages ( Figures 1E-ii,iii ). As a control, lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), the vital component for speckle structure in the nucleus that might participate in the viral life cycle ( Swain et al, 2021 ), was detected, and the data showed that MALAT1 expression increased from 0 to 24 hpi and then collapsed ( Figure 1F ). Intriguingly, the transcription level of NEAT1-2 seemed to persistently exhibit higher fold changes in macrophages than in monocytes ( Figure 1E-iii ), and macrophages maintained a relatively lower viral load of HTNV than their counterpart monocytes ( Figure 1G ).…”