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Functional analysis of the Arabidopsis thaliana Pol V largest subunit, NRPE1, carboxy-terminal domain by whole genome bisulfite sequencing

ABSTRACT: RNA Polymerase V transcription recruits siRNA-Argonaute protein complexes to chromatin, thereby specifying sites of RNA-directed DNA methylation (RdDM) and transcriptional gene silencing in plants. The Pol V largest subunit, NRPE1, has an extensive carboxyl-terminal domain (CTD) that is dispensable for catalytic activity in vitro, yet essential in vivo. A CTD subdomain, DeCL, named for its similarity to a chloroplast protein, DEFECTIVE CHLOROPLASTS AND LEAVES, is required for Pol V function at virtually all loci, similar to mutants defective for Pol V recruitment. Deletions removing three other CTD subdomains affect overlapping subsets of loci, similar to mutants lacking proteins that bind Pol V or its transcripts. A yeast two-hybrid screen for CTD-interactors identified the 3-prime -> 5-prime exoribonuclease, RRP6L1 as an interactor with the DeCL subdomain and the adjacent QS subdomain, named for its numerous glutamine-serine (QS) repeats. These RRP6L1-binding subdomains immediately follow the Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3-prime ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the adjacent CTD subdomains enable the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE93358 | GEO | 2017/02/25

SECONDARY ACCESSION(S): PRJNA360690

REPOSITORIES: GEO

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3'RACE analyses of Pol V transcript 3' end lengths in mutants of the exonuclease RRP6L1

Project description:RNA Polymerase V transcription recruits siRNA-Argonaute protein complexes to chromatin, thereby specifying sites of RNA-directed DNA methylation (RdDM) and transcriptional gene silencing in plants. The Pol V largest subunit, NRPE1, has an extensive carboxyl-terminal domain (CTD) that is dispensable for catalytic activity in vitro, yet essential in vivo. A CTD subdomain, DeCL, named for its similarity to a chloroplast protein, DEFECTIVE CHLOROPLASTS AND LEAVES, is required for Pol V function at virtually all loci, similar to mutants defective for Pol V recruitment. Deletions removing three other CTD subdomains affect overlapping subsets of loci, similar to mutants lacking proteins that bind Pol V or its transcripts. A yeast two-hybrid screen for CTD-interactors identified the 3-prime -> 5-prime exoribonuclease, RRP6L1 as an interactor with the DeCL subdomain and the adjacent QS subdomain, named for its numerous glutamine-serine (QS) repeats. These RRP6L1-binding subdomains immediately follow the Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3-prime ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the adjacent CTD subdomains enable the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

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