Abstract
Plants employ five DNA-dependent RNA polymerases (Pols) in transcription. One of these polymerases, Pol III, has previously been reported to transcribe 5S rRNA, tRNAs, and a number of small RNAs. However, in-depth functional analysis is complicated by the fact that knockout mutations in Pol subunits are typically lethal. Here, we report the characterization of the first known viable Pol III subunit mutant,nrpc7-1 This mutant was originally isolated from a forward genetic screen designed to identify enhancers of the autoimmune mutantsnc1, which contains a gain-of-function mutation in a nucleotide-binding leucine-rich repeat (NLR) immune receptor-encoding gene. Thenrpc7-1mutation occurs in an intron-exon splice site and results in intron retention in someNRPC7transcripts. There is a global disruption in RNA equilibrium innrpc7-1, exemplified by the altered expression of a number of RNA molecules, some of which are not reported to be transcribed by Pol III. There are developmental defects associated with the mutation, as homozygous mutant plants are dwarf, have stunted roots and siliques, and possess serrated leaves. These defects are possibly due to altered small RNA stability or activity. Additionally, thenrpc7-1mutation confers anNLR-specific alternative splicing defect that correlates with enhanced disease resistance, highlighting the importance of alternative splicing in regulating NLR activity. Altogether, these results reveal novel roles for Pol III in maintaining RNA homeostasis, adjusting the expression of a diverse suite of genes, and indirectly modulating gene splicing. Future analyses using thenrpc7-1mutant will be instrumental in examining other unknown Pol III functions.
Highlights
Transcription under both static and dynamic conditions requires the action of evolutionarily conserved multi-subunit enzymes known as DNA-dependent RNA polymerases (Pols)
To avoid potential lethality resulting from dramatically enhanced autoimmunity, the snc1 suppressor mos4 was included in the genetic background of the screen
Seeds from the wild type-like mos4 snc1 plants were mutagenized with ethyl methanesulfonate, and the M2 population was screened for plants displaying a reversion to snc1-like morphology and resistance
Summary
Transcription under both static and dynamic conditions requires the action of evolutionarily conserved multi-subunit enzymes known as DNA-dependent RNA polymerases (Pols). All eukaryotes possess three distinct RNA polymerases (Pols I, II, and III), each of which transcribes specific suites of genes (Cramer et al, 2008). Pol I transcribes 45S rRNA, which is the precursor to 5.8S, 18S and 25S rRNAs. Pol II transcribes mRNAs as well as most small nuclear (sn)RNAs and micro (mi)RNAs. Pol III was previously thought to be primarily required for the transcription of ‘housekeeping’ genes such as those encoding 5S rRNA and tRNAs. recent reports indicate that the
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