Functions of Arabidopsis C-terminal Domain Phosphatase-like 4 in Global Transcriptional Regulation

Phosphoregulation of the carboxyl-terminal domain of RNA polymerase II largest subunit (pol II-CTD) couples transcription and co-transcriptional modification of nascent RNA. Although the molecular mechanisms have been extensively studied in vertebrates, understanding of that in plants is still in its infancy. Through genetics, biochemical and transcriptomic approaches, this dissertation work characterizes functions of a pol II-CTD phosphatase-like protein from Arabidopsis thaliana, CPL4, in the phosphoregulation of pol II-CTD during protein-coding and non-coding RNA transcriptions. CPL4 interacts with and dephosphorylates pol II-CTD both in vitro and in vivo, showing that CPL4 regulates pol II-CTD phosphorylation status in Arabidopsis. An amino acid substitution in the catalytic motif abolished the phosphatase activity of CPL4. The catalytically inactive protein strongly inhibits transcription in transient assays, likely due to a dominant negative effect. Deletion of Breast cancer C-terminal (BRCT) domain alleviates the inhibitory effect of the catalytically inactive CPL4, suggesting that BRCT domain is necessary for CPL4’s function. A suite of xenobiotic stress responsive genes shows constitutive up-regulation in CPL4 knockdown transgenic (CPL4RNAi) lines, indicating that CPL4 negatively regulates the toxic chemical detoxification pathway. The CPL4RNAi plants accumulate aberrant 3’-extended transcripts from many pol II-dependent small nuclear RNA (snRNA) loci. The snRNA 3’-extension gives rise to a snRNA transcript fused with a downstream protein-coding gene (DPG) if present. Such snRNA-DPG fusion transcripts can be found in other plant species. A short, unstable non-coding RNA produced from a protein-coding locus driven by a transposable-embedded snRNA promoter can yield the full-length product in the CPL4RNAi plants. These snRNA-DPGs can be induced in wild type by salt stress, which affects pol II-CTD phosphorylation status. These results indicate a potential stress-inducible conversion of non-coding RNA transcription into protein-coding transcription mediated by pol II-CTD phosphoregulation. CPL4RNAi root explants exhibit enhanced capability of de novo shoot organogenesis due to cytokinin hypersensitivity and earlier induction of shoot apical meristem regulatory genes. A potential involvement of an operon-like cluster of thalianol biosynthesis genes in the CPL4RNAi organogenesis phenotype is implicated. Taken together, Arabidopsis CPL4 is an essential pol II-CTD phosphatase, regulating stress-responsive and organogenesis pathways through protein-coding and non-coding RNA transcriptions

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