Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency

Andrea Graziadei, Pawel Masiewicz, Audrone Lapinaite, Teresa Carlomagno

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2′-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2′-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA from Pyrococcus furiosus. We find that the methylation efficacy at sites D and D′ differ substantially, with substrate D′ turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D′ is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D′ sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5′-end of the product.

Original languageEnglish (US)
Pages (from-to)764-772
Number of pages9
JournalRNA
Volume22
Issue number5
DOIs
StatePublished - May 2016

Keywords

  • 2′-O methylation
  • Box C/D RNP
  • RNA modification
  • Regulatory mechanism

ASJC Scopus subject areas

  • Molecular Biology

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