Elucidating microRNA-34a organisation within human Argonaute-2 by dynamic nuclear polarisation-enhanced magic angle spinning NMR.
Rubin DasguptaWalter BeckerKatja PetzoldPublished in: Nucleic acids research (2024)
Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, which is de-regulated in various cancers, in human Argonaute-2 (hAgo2), the effector protein in RISC. This analysis employs guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced Magic Angle Spinning (MAS) NMR. Homonuclear correlation experiments revealed that the non-A-form helical conformation of miR-34a increases when incorporated into hAgo2 and subsequently bound to SIRT1 mRNA compared to the free miR-34a or the free mRNA:miR duplex. The C8-C1' correlation provided a nucleotide-specific distribution of C2'- and C3'-endo sugar puckering, revealing the capture of diverse dynamic conformations upon freezing. Predominantly C3'-endo puckering was observed for the seed region, while C2'-endo conformation was found in the central region, with a mixture of both conformations elsewhere. These observations provide insights into the molecular dynamics underlying miR-mediated mRNA regulation and demonstrate that experiments conducted under cryogenic conditions, such as at 90 K, can capture and reveal frozen dynamic states, using methods like DNP-enhanced MAS NMR or Cryo-Electron Microscopy.
Keyphrases
- cell proliferation
- long non coding rna
- long noncoding rna
- high resolution
- solid state
- molecular dynamics
- magnetic resonance
- endothelial cells
- electron microscopy
- binding protein
- oxidative stress
- transcription factor
- gene expression
- dendritic cells
- regulatory t cells
- density functional theory
- immune response
- high glucose
- young adults
- ischemia reperfusion injury