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DNAzyme-dependent Analysis of rRNA 2’-O-Methylation
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A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation.

Andrew Loffer1, Jasleen Singh1, Akihito Fukudome1,2

  • 1Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University Bloomington, Bloomington, United States.

Elife
|January 31, 2022
PubMed
Summary

Plant RNA-directed DNA methylation relies on small interfering RNAs (siRNAs) generated by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Their combined activities create a code influencing siRNA size and precursor processing for effective gene silencing.

Keywords:
A. thalianaRNA polymeraseRNA silencingRNA-directed DNA methylationchromosomesdicer endonucleasedicer-like 3gene expressiongeneticsgenomicssiRNA biogenesis

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Area of Science:

  • Plant molecular biology
  • Epigenetics
  • RNA interference

Background:

  • Selfish genetic elements in plants are silenced via RNA-directed DNA methylation.
  • Short interfering RNAs (siRNAs) guide this silencing process.
  • DICER-LIKE 3 (DCL3) processes double-stranded RNA precursors into siRNAs.

Purpose of the Study:

  • To investigate how NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) influence siRNA generation.
  • To understand the mechanisms determining siRNA size and precursor processing.
  • To elucidate how these factors contribute to maximal siRNA coverage at target loci.

Main Methods:

  • Analysis of nucleotide initiation by Pol IV.
  • Characterization of RDR2 initiation relative to Pol IV transcripts.
  • Assessment of RDR2 terminal transferase activity.
  • Examination of DCL3 dicing patterns.

Main Results:

  • Pol IV initiation nucleotide choice impacts precursor processing.
  • RDR2 initiation site and terminal transferase activity generate a code.
  • This code dictates which precursor end is diced and siRNA size (24 or 23 nt).
  • Alternative DCL3 dicing patterns diversify siRNA characteristics.

Conclusions:

  • A nucleotide-based code governs siRNA production from Pol IV/RDR2 precursors.
  • This code optimizes siRNA size, sequence, and strand specificity.
  • Diversified siRNAs ensure comprehensive epigenetic silencing of target loci.