Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana

Okanami, Masahiro; Meshi, Tetsuo; Iwabuchi, Masaki

doi:10.1093/nar/26.11.2638

Cited by 51 publications

(29 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The amplified DNA was fractionated on a 1.2% agarose gel and transferred to nylon membranes (Sambrook et al, 1989). In the case of semiquantitative reverse transcriptase-mediated PCR to analyze AtCDC6 expression in the transgenic lines, the blots were prehybridized and hybridized using perfectHyb buffer (Sigma) and radiolabeled AtCDC6 or AtDHR1 probe (Okanami et al, 1998;Castellano et al, 2001). …”

Section: Dna and Rna Manipulationmentioning

confidence: 99%

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCF^AtSKP2 Pathway in Response to Light

2002

View full text Add to dashboard Cite

Selective ubiquitin-mediated proteolysis through the cell cycle controls the availability, and therefore the activity, of several cell proliferation proteins. E2F transcription factors play distinct roles in both proliferating and differentiated cells by regulating gene expression. Here, we report that Arabidopsis AtE2Fc is regulated by a balance between gene expression and ubiquitin-proteasome proteolysis. AtE2Fc degradation implicates the function of the E3 ubiquitin-ligase Skp1, Cullin, F-box (SCF AtSKP2 ) complex and seems to be dependent on cyclin-dependent kinase phosphorylation. In addition, we found that AtE2Fc degradation is triggered by light stimulation of dark-grown seedlings. Interestingly, the auxin response mutant axr1-12 , in which RUB1 modification of the SCF component CUL1 is impaired, shows increased AtE2Fc protein levels, suggesting a dysfunction in the control of AtE2Fc stability. Likewise, overexpression of a stable form of the AtE2Fc protein negatively affects cell division and increases cell size. These effects are mediated, at least in part, by downregulating the cell cycle gene AtCDC6 . The negative role of AtE2Fc in gene expression is further supported by the fact that AtE2Fc interacts with plant retinoblastoma-related protein, suggesting that AtE2Fc might form part of a repressor complex. We propose that AtE2Fc might play a role in cell division and during the transition from skotomorphogenesis to photomorphogenesis.

show abstract

Section: Dna and Rna Manipulationmentioning

confidence: 99%

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCF^AtSKP2 Pathway in Response to Light

2002

View full text Add to dashboard Cite

show abstract

“…Surprisingly any of the other NTPs or dNTPs can substitute for ATP in the editing reaction, which is not very common for enzymatically catalyzed reactions. Some of the few enzymes accepting NTP or dNTP belong to a subgroup of the RNA helicases (24,25). RNA helicases have been identified in plants through in silico analyses of the genomic and cDNA data in the databases and a number of putative genes have been found in the A. thaliana genome (26).…”

Section: Figmentioning

confidence: 99%

In Vitro RNA Editing in Pea Mitochondria Requires NTP or dNTP, Suggesting Involvement of an RNA Helicase

Takenaka

Brennicke

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn 2؉ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mM. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.RNA editing has been observed in various forms in diverse organisms including trypanosomes, mammals, slime molds, and land plants (1). In land plants, RNA editing has so far been reported in mitochondria and plastids. In flowering plants the majority of this organellar editing involves C to U conversions, with U to C changes occurring much more infrequently and only in mitochondria (2, 3, 4). In non-flowering plants this latter direction of RNA editing is much more common in mitochondria as well as in chloroplasts, the U to C reactions almost reaching the frequency of the C to U editing in the hornworts (5). Since its discovery more than a decade ago, progress into understanding how this process works has been hampered by the lack of manageable and reliable in vitro systems. The first in vitro system for plant mitochondrial RNA editing was successfully developed from wheat embryos (6), but has not been exploited further.Conclusions about the determinants of specificity during RNA editing in mitochondria and in plastids have initially been drawn from comparisons of transcribed sequence duplications (e.g. 7). Such comparisons suggested that upstream (5Ј) sequences are crucial in targeting the editing machinery and that downstream similarities are not sufficient to specify a site.These observations were corroborated by the recent breakthrough development of an in vitro editing system for plastids (8, 9) and an electroporation protocol for mitochondria (10,11). Mutational analysis of templates in these as well as in in vivo experiments in transgenic chloroplasts (12)(13)(14) showed that for several editing sites 20 -30 nucleotides upstream and 2-5 nucleotides downstream are sufficient to guide the editing activity, precisely what had initially been deduced for mitochondria (7).The biochemistry of this RNA editing activity is still largely unclear, and more detailed u...

show abstract

“…Some viral helicases, such as NPHII and HCV NS3, also have DNA-DNA and RNA-RNA helicase activities (6,28,29). In the case of DEAD box proteins, DP103 and AtDRH1 show dsRNA unwinding and DNA-RNA helicase activities (30,31), but no DEAD box protein has been shown to have DNA helicase activity. The DNA helicase activity of Dbp9p was found to be abolished by an amino acid substitution in its RNA-binding domain (Fig.…”

mentioning

confidence: 99%

Dbp9p, a Member of the DEAD Box Protein Family, Exhibits DNA Helicase Activity

Kikuma

Ohtsu

Utsugi

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.RNA helicases are enzymes that unwind double-stranded RNA molecules in an energy-dependent manner through the hydrolysis of NTP. These proteins are widely distributed among a variety of organisms ranging from viruses and prokaryotes to mammals. RNA helicases are associated with virtually all biological processes requiring RNA, including transcription, splicing, RNA transport, ribosome biogenesis, RNA editing, translation, and RNA decay (1). The largest family of RNA helicases is the DEAD box protein family.The DEAD box proteins have seven to eight distinctive motifs. The DEAD is derived from the amino acid sequence of motif II, the Walker B motif (2, 3). In vitro analyses of DEAD box proteins such as the translation initiation factor eIF-4A and the human nuclear protein p68 have demonstrated that these proteins possess RNA-dependent ATPase activity and are capable of melting short RNA duplex structures in an ATP-dependent manner (3-5). For example, eIF-4A, an archetypical member of the DEAD box protein family, is capable of unwinding partial duplex RNA in a bidirectional manner and acting on RNA or DNA-RNA, but not on the DNA duplex (4, 6). Extensive mutational analyses of the conserved regions of DEAD box proteins have demonstrated that these regions are important to ATP binding, ATP hydrolysis, RNA binding, RNA unwinding, and coupling of these different activities. In addition to these typical DEAD box RNA helicases, some DEAD box proteins have recently been shown to have peculiar characteristics. Hepatitis C virus NS3 drives the unwinding activity with all ribo-and deoxyribo-NTPs (6). Moreover, CsdA, an Escherichia coli DEAD box protein, unwinds double-stranded RNA in the absence of NTP (7). These reports suggest that the DEAD box family includes proteins with a wide variety of biochemical activities.The yeast Saccharomyces cerevisiae contains over 20 different DEAD box proteins, many of which are essential to viability. Combined with biochemical analyses, yeast genetical analyses have revealed the functions of many DEAD box proteins. ...

show abstract

Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana

Cited by 51 publications

References 32 publications

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCF^AtSKP2 Pathway in Response to Light

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCF^AtSKP2 Pathway in Response to Light

In Vitro RNA Editing in Pea Mitochondria Requires NTP or dNTP, Suggesting Involvement of an RNA Helicase

Dbp9p, a Member of the DEAD Box Protein Family, Exhibits DNA Helicase Activity

Contact Info

Product

Resources

About

Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana

Cited by 51 publications

References 32 publications

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCFAtSKP2 Pathway in Response to Light

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCFAtSKP2 Pathway in Response to Light

In Vitro RNA Editing in Pea Mitochondria Requires NTP or dNTP, Suggesting Involvement of an RNA Helicase

Dbp9p, a Member of the DEAD Box Protein Family, Exhibits DNA Helicase Activity

Contact Info

Product

Resources

About

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCF^AtSKP2 Pathway in Response to Light

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCF^AtSKP2 Pathway in Response to Light