Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation

Jered M Wendte; Jeremy R Haag; Jasleen Singh; Anastasia McKinlay; Olga M Pontes; Craig S Pikaard

doi:10.1016/j.celrep.2017.05.091

Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation

Cell Rep. 2017 Jun 27;19(13):2796-2808. doi: 10.1016/j.celrep.2017.05.091.

Authors

Jered M Wendte¹, Jeremy R Haag², Jasleen Singh¹, Anastasia McKinlay¹, Olga M Pontes³, Craig S Pikaard⁴

Affiliations

¹ Department of Biology, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA.
² Department of Biology, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA; Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63130, USA.
³ Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63130, USA.
⁴ Department of Biology, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA; Department of Molecular and Cellular Biochemistry, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA; Howard Hughes Medical Institute, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA. Electronic address: cpikaard@indiana.edu.

Abstract

Plant multisubunit RNA polymerase V (Pol V) transcription recruits Argonaute-small interfering RNA (siRNA) complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive C-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro yet essential in vivo. One CTD subdomain (DeCL) is required for Pol V function at virtually all loci. Other CTD subdomains have locus-specific effects. In a yeast two-hybrid screen, the 3'→ 5' exoribonuclease RRP6L1 was identified as an interactor with the DeCL and glutamine-serine (QS)-rich subdomains located downstream of an Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3' ends of Pol V transcripts sliced by Argonaute 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

Keywords: Pol V transcription; chromatin modification; cytosine methylation; gene silencing; noncoding RNA; siRNA.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

DNA Methylation / immunology*
DNA-Directed RNA Polymerases / immunology*
Gene Silencing / immunology*

Substances

DNA-Directed RNA Polymerases

Abstract

Publication types

MeSH terms

Substances

Grants and funding