Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

Caijie Zhao, Ravichandra Bachu, Milena Popović, Matthew Devany, Michael Brenowitz, Jörg C. Schlatterer, Nancy L. Greenbaum

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

The complex formed between the U2 and U6 small nuclear (sn)RNA molecules of the eukaryotic spliceosome plays a critical role in the catalysis of precursor mRNA splicing. Here, we have used enzymatic structure probing, 19F NMR, and analytical ultracentrifugation techniques to characterize the fold of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex. Results from enzymatic probing and 19F NMR for the complex in the absence of Mg2+ are consistent with formation of a four-helix junction structure as a predominant conformation. However, 19F NMR data also identify a lesser fraction (up to 14% at 25°C) of a three-helix conformation. Based upon this distribution, the calculated ΔG for interconversion to the four-helix structure from the three-helix structure is approximately -4.6 kJ/mol. In the presence of 5 mM Mg2+, the fraction of the three-helix conformation increased to ∼17% and the Stokes radius, measured by analytical ultracentrifugation, decreased by 2%, suggesting a slight shift to an alternative conformation. NMR measurements demonstrated that addition of an intron fragment to the U2-U6 snRNA complex results in displacement of U6 snRNA from the region of Helix III immediately 5′ of the ACAGAGA sequence of U6 snRNA, which may facilitate binding of the segment of the intron adjacent to the 5′ splice site to the ACAGAGA sequence. Taken together, these observations indicate conformational heterogeneity in the protein-free human U2-U6 snRNA complex consistent with a model in which the RNA has sufficient conformational flexibility to facilitate inter-conversion between steps of splicing in situ.

Original languageEnglish (US)
Pages (from-to)561-573
Number of pages13
JournalRNA
Volume19
Issue number4
DOIs
StatePublished - Apr 1 2013

Keywords

  • Conformational heterogeneity
  • Enzymatic structure probing
  • F-NMR
  • Secondary structure
  • Spliceosome
  • U2-U6 snRNA

ASJC Scopus subject areas

  • Molecular Biology

Fingerprint Dive into the research topics of 'Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex'. Together they form a unique fingerprint.

  • Cite this

    Zhao, C., Bachu, R., Popović, M., Devany, M., Brenowitz, M., Schlatterer, J. C., & Greenbaum, N. L. (2013). Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex. RNA, 19(4), 561-573. https://doi.org/10.1261/rna.038265.113