Role of RNA polymerase IV in plant small RNA metabolism

doi:10.1073/pnas.0611456104

Role of RNA polymerase IV in plant small RNA metabolism

^†Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095;
^‡Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711; and
^§Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095

Edited by Susan R. Wessler, University of Georgia, Athens, GA, and approved January 22, 2007 (received for review December 22, 2006)

Abstract

In addition to the three RNA polymerases (RNAP I–III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced ≈335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors.

Footnotes

^¶To whom correspondence should be addressed. E-mail: jacobsen{at}ucla.edu
Author contributions: X.Z. and S.E.J. designed research; X.Z., I.R.H., and C.L. performed research; P.J.G. contributed new reagents/analytic tools; X.Z. analyzed data; X.Z. wrote the paper; and S.E.J. edited the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0611456104/DC1.
Abbreviations:

sRNA,

small RNA;

miRNA,

microRNA;

tasiRNA,

transacting siRNA;

nat-siRNA,

natural–antisense siRNA;

RNAP,

RNA polymerase;

PM,

perfectly matched Arabidopsis sequences;

MPSS,

massively parallel signature sequencing;

RISC,

RNA-induced silencing complex;

RDR2,

RNA-dependent RNAP 2.
Freely available online through the PNAS open access option.