A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase

Jost, Jean-Pierre; Schwarz, Steffen; Heß, Daniel; Angliker, Herbert; Fuller-Pace, Frances V.; Stahl, Hans; Thiry, Stéphane; Siegmann, Michel

doi:10.1093/nar/27.16.3245

Cited by 59 publications

(37 citation statements)

References 46 publications

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“…The data suggest that p68 RNA helicase plays important roles in normal cell growth. Although, p68 RNA helicase was suspected of being involved in a number of biological processes in cells (8,12,17), the experimental evidence that links p68 RNA helicase to a specific cellular process is still 5 to 7). The extracts were pretreated with either RNase H and DNA oligonucleotide ␣U1 1-13 (lanes 3, 4, 6, and 7) or no RNase H treatment (lane 2 and 5).…”

Section: Discussionmentioning

confidence: 99%

p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

Liu

2002

Molecular and Cellular Biology

127

102

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Modulation of the interaction between U1 snRNP and the 5 splice site (5ss) is a key event that governs 5ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5ss during early stage of splicing. However, dissociation of the U1 from the 5ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.To remove introns, a large RNA-protein complex, known as the spliceosome, must be assembled on mRNA precursors (pre-mRNA) (11,29,39). Assembly of a functional spliceosome proceeds through an ordered addition of four small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4/U6, and U5) as well as many non-snRNP proteins. This pathway leads to the formation of several intermediate spliceosome complexes. Recognition of the 5Ј splice site (5Јss) by the U1 snRNP, along with binding of the polypyrimidine tract and branch point by U2AF65 and SF1, results in the formation of the commitment complex. Recruitment of the U2 snRNP to the commitment complex leads to the formation of complex A, or the prespliceosome. At this point, the preformed U4/U6.U5 tri-snRNPs will join into the prespliceosome, leading to the formation of the spliceosome (1, 13, 28).The spliceosome is a dynamic molecular machine. Dynamic RNA-RNA base-pairing interactions between snRNAs and pre-mRNA and among snRNAs play critical roles in the spliceosome assembly and recognition of splice sites (32,33,35). The RNA duplexes in the spliceosome complexes are generally transient and short, ranging from 3 to 7 bp. Many of these RNA-RNA interactions are mutually exclusive. One example is the RNA-RNA interactions that occur at the 5Јss. The 5Јss is first recognized by interactions of 5 to 7 bp between the 5Јss and 5Ј-end of the U1 snRNA. This early U1-5Јss duplex is unwound to expose the same 5Јss sequence for pairing with the U6 snRNA prior to the first chemical reaction of splicing (28). The U6 snRNA is associated with U4 by two RNA helices before joining the prespliceosome. The two RNA helices between U6 and U4 must be unwound before U6 pairing with the 5Јss (2, 30, 40). Three events, unwinding of the U1-5Јss duplex, unwinding of the U4/U6 helices, and formation of the U6-5Јss duplex, are tightly coupled. How does the spliceosome coordinate these RNA-RNA interactions precisely in time and space? Presumably, protein factors are ne...

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Section: Discussionmentioning

confidence: 99%

p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

Liu

2002

Molecular and Cellular Biology

127

102

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“…Both proteins may therefore participate in structural reorganization of ribonucleoprotein assemblies in vivo. In fact, p68 has been found in spliceosomes (15) and in a protein-RNA complex of 5-methylcytosine-DNA glycosylase (16). A recently reported (17) interaction with protein kinase A anchoring protein AKAP95 supports a role of p68 in hormonally responsive transcription complexes, and indeed, p68 and p72 act as transcriptional co-activators of estrogen receptor ␣ in cooperation with an RNA co-activator (18,19).…”

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confidence: 89%

The mRNA of DEAD Box Protein p72 Is Alternatively Translated into an 82-kDa RNA Helicase

Uhlmann‐Schiffler

Rößler

Stahl

2002

Journal of Biological Chemistry

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p68 and p72 are two highly related DEAD box proteins with similar biochemical activities in the nucleus of vertebrate cells; it is unknown whether they have redundant or differential in vivo functions. We report on a third member of this subfamily that is alternatively expressed from p72 mRNA. A detailed analysis of HeLa p72 mRNA was performed. It has an overall length of more than 5 kb and contains a 0.75-kb 5-untranslated region and a 3-untranslated region of 2.5 kb. Its open reading frame extends to nucleotide ؊243 upstream of the first in-frame AUG (A in the AUG triplet is ؉1) which serves as the p72 translation initiator codon. We provide evidence that alternative translation at a non-AUG within the extra coding region of this mRNA yields an 82-kDa protein (p82). Immunological studies substantiate that p82 is a naturally existing p72 variant and that both proteins are expressed at similar concentrations. p82 purified from HeLa cells is an ATP-dependent RNA helicase with biochemical properties almost identical to those of p72.Control of transcription is generally considered the main regulation level of mammalian gene expression, although mRNA maturation and modulation of mRNA stability or translational efficiency also contribute to expression control (1). Generation of protein variants by alternative pre-mRNA splicing or alternative initiation of mRNA translation enlarges the coding capacity and may explain the relatively low number of genes actually found in the human genome. According to recent estimations, on average one gene encodes three proteins (2).Although the 5Ј-untranslated leader sequences (UTRs) 1 of most vertebrate mRNAs are less than 100 nucleotides (nt) long (3), those of tightly controlled genes usually are longer and are GC-rich. Such structure-prone 5Ј-UTRs can modulate translation by the presence of upstream (up) open reading frames (ORFs) in addition to the main ORF, by formation of secondary structures, and/or by RNA-protein interactions (4). Indeed, they appear particularly characteristic of mRNAs encoding growth factors, receptor proteins, transcription factors, signal transduction components, proto-oncogenes, tumor suppressor proteins, and even proteins that mostly are constitutively expressed at a low level ("housekeeping" proteins).DEAD/DEXH box proteins belonging to superfamily II of RNA helicases play a universal role in RNA metabolism of proand eukaryotes. Not surprisingly, they are involved in gene expression control, for instance during germ cell and embryonic development or in cell proliferation and differentiation (5). This in turn requires tight control of at least some DEAD/DEXH box proteins like CsdA of Escherichia coli, which is regulated by an 11-nt "cold box" sequence in its 5Ј-UTR (6), or the Saccharomyces cerevisiae Dbp2, the gene of which contains an unusually large intron that is part of a post-transcriptional autoregulatory feedback loop (7). A similarly complex transcription regulation has been shown for nuclear p68 (8), a mammalian homologue of Dbp2. In addition,...

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“…Earlier work has shown that 5-methylcytosine was replaced by labeled cytosine during the demethylation reaction in erythroleukemia cells, indicating a replacement of the entire nucleotide or base alone (9). One potential mechanism to achieve this is through the action of 5-methylcytosine DNA glycosylase, which removes the methylcytosine from DNA leaving the deoxyribose intact (10). Local DNA repair then removes the abasic nucleotide and adds back an unmethylated cytosine nucleotide (11).…”

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confidence: 99%

Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation

Agius

Kapoor

Zhu

2006

Proc. Natl. Acad. Sci. U.S.A.

279

239

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DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive ␤-and ␦-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase͞lyase important for active DNA demethylation in Arabidopsis.DNA methylation ͉ epigenetics ͉ transcriptional gene silencing D NA cytosine methylation is important for many epigenetic processes including X chromosome inactivation, genomic imprinting, epigenetic changes during carcinogenesis, and silencing of transposons, of specific genes during development and of certain transgenes (1-5). The enzymes responsible for de novo as well as maintenance methylation at the 5Ј position of cytosines have been well characterized, and mutations in these enzymes can release transcriptional gene silencing and cause various developmental phenotypes (2,3,6,7). In contrast, the mechanism of DNA demethylation is less understood. DNA demethylation can be passive or active. Passive demethylation occurs automatically for newly synthesized DNA during replication if the new DNA is not acted upon by DNA methyltransferases. The biochemical mechanism of active DNA demethylation has been controversial (8).The chemistry of demethylating 5-methylcytosine DNA is challenging because it requires the disruption of carbon-carbon bonds. Earlier work has shown that 5-methylcytosine was replaced by labeled cytosine during the demethylation reaction in erythroleukemia cells, indicating a replacement of the entire nucleotide or base alone (9). One potential mechanism to achieve this is through the action of 5-methylcytosine DNA glycosylase, which removes the methylcytosine from DNA leaving the deoxyribose intact (10). Local DNA repair then removes the abasic nucleotide and adds back an unmethylated cytosine nucleotide (11). Using chicken embryo nuclear extracts that can promote demethylation (12), a putative demethylase was purified; this was found to be a G͞T mismatch repair DNA glycosylase (13). MBD4, a human homolog of the chicken enzyme, also has 5-methylcytosine DNA glycosylase activity (14). At least in vitro, these G͞T mismatch repair ...

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A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase

Cited by 59 publications

References 46 publications

p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

The mRNA of DEAD Box Protein p72 Is Alternatively Translated into an 82-kDa RNA Helicase

Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation

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