Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation (original) (raw)
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The Plant journal : for cell and molecular biology, 2017
The evolutionarily conserved 12-subunit RNA polymerase II (Pol II) is a central catalytic component that drives RNA synthesis during the transcription cycle that consists of transcription initiation, elongation, and termination. A diverse set of general transcription factors, including a multifunctional TFIIF, govern Pol II selectivity, kinetic properties, and transcription coupling with posttranscriptional processes. Here, we show that TFIIF of Arabidopsis (Arabidopsis thaliana) resembles the metazoan complex that is composed of the TFIIFα and TFIIFβ polypeptides. Arabidopsis has two TFIIFβ subunits, of which TFIIFβ1/MAN1 is essential and TFIIFβ2/MAN2 is not. In the partial loss-of-function mutant allele man1-1, the winged helix domain of Arabidopsis TFIIFβ1/MAN1 was dispensable for plant viability, whereas the cellular organization of the shoot and root apical meristems were abnormal. Forward genetic screening identified an epistatic interaction between the largest Pol II subunit ...
Proteomics, 2014
The elongation phase of the RNA polymerase II (RNAPII) transcription process is dynamic and regulated. Elongator and SUPPRESSOR OF Ty4 (SPT4)/SPT5 are transcript elongation factors that contribute to the regulation of mRNA synthesis by RNA polymerase II in the chromatin context. Recently, the Elongator complex consisting of six subunits and the SPT4/SPT5 heterodimer were isolated from Arabidopsis. Mutant plants affected in the expression of Elongator or SPT4/SPT5 share various auxin-signaling phenotypes. In line with that observation, auxin-related genes are prominent among the genes differentially expressed in these mutants. Exemplified by Elongator and SPT4/SPT5, we discuss here the role that transcript elongation factors may play in the control of plant growth and development.
Transcript Elongation Factor TFIIS Is Involved in Arabidopsis Seed Dormancy
Journal of Molecular Biology, 2009
Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS −/− plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.
Genes & Development, 2005
Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production.
Protoplasma, 2014
The mechanisms of plant cell dedifferentiation and the acquisition of totipotency are poorly understood. One of the methods to induce the dedifferentiation process in plant cells is simple and requires the removal of the cell wall. After cell wall removal in protoplasts, large-scale chromatin decondensation is observed (Tessadori et al. in J Cell Sci 120:1200-1208. Here, we show that in Arabidopsis thaliana protoplasts, despite chromatin decondensation, RNA polymerase II transcriptional activity is reduced. The subsequent investigated stages displayed a clear decrease in the quantity of 25S ribosomal RNA (rRNA) first and then poly(A+) RNA, particularly in the cytoplasm. Therefore, the reduced transcription activity and the removal of these RNA transcripts from the cytoplasm is a crucial process in obtaining totipotency in plant cells. After the cytoplasm cleaning of transcripts derived from mesophyll cells, we observed the resynthesis of these RNAs. An increase in the amount of examined molecules to a level similar to that in differentiated mesophyll cells precedes the divisions of already undifferentiated cells. In this work, we show changes in RNA polymerase II transcription dynamics and the quantity of poly(A+) RNA and 25S rRNA during dedifferentiation and re-entry into the cell cycle.
Proceedings of The National Academy of Sciences, 1994
RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells. At least one protein, THIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through. The C-terminal halfofthe TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro. To study how TFHIS changes the response of RNA polymerase II elongation complexes to such blocks, targeted amino acids in the C terminus of HeLa TFIIS were mutated to alanines. Two mutant TFIIS proteins as well as the unutated C-terminal half of the TFUIS protein were purified following overexpression in Escherichia colH. Each protein was examined for read-through activity and ability to stimulate transcript cleavage in ternary elongation complexes. Mutant TFIIS5 (E174A, E175A) was reduced in read-through and cleavage activities relative to the unmutated, truncated TFHIS (ATFIIS). Mutant TFIIS7 (K187A, K189A) was able to stimulate cleavage nearly at the rate and to the extent of the TFIIS5 mutant. In contrast to what
Molecular Cell, 2010
During transcript elongation in vitro, backtracking of RNA polymerase II (RNAPII) is a frequent occurrence that can lead to transcriptional arrest. The polymerase active site can cleave the transcript during such backtracking, allowing transcription to resume. Transcript cleavage is either stimulated by elongation factor TFIIS or occurs much more slowly in its absence. However, whether backtracking actually occurs in vivo, and whether transcript cleavage is important to escape it, has been unclear. Using a yeast TFIIS mutant that lacks transcript cleavage stimulatory activity and simultaneously inhibits unstimulated cleavage, we now provide evidence that escape from backtracking via transcript cleavage is essential for cell viability and efficient transcript elongation. Our results suggest that transcription problems leading to backtracking are frequent in vivo and that reactivation of backtracked RNAPII is crucial for transcription.
Journal of experimental botany, 2016
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 mutant snc1, which contains a gain-of-function mutation in a nucleotide-binding leucine-rich repeat (NLR) immune receptor-encoding gene. The nrpc7-1 mutation occurs in an intron-exon splice site and results in intron retention in some NRPC7 transcripts. There is a global disruption in RNA equilibrium in nrpc7-1, 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 developmen...