The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells

Ogilvie, Varrie; Wilson, B. J.; Nicol, Samantha M.; Morrice, Nick; Saunders, Laura R.; Barber, Glen N.; Fuller-Pace, FV

doi:10.1093/nar/gkg236

Cited by 78 publications

(85 citation statements)

References 38 publications

(62 reference statements)

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“…p72 could be a candidate for this redundant function. It was recently demonstrated that p68 and p72 exist as a heterodimer in cells (34). Furthermore, p72 was shown to associate with the U1 snRNP (24), and the protein plays a role in regulating the splicing of alternative exons containing AC-rich exon enhancer elements (15).…”

Section: Discussionmentioning

confidence: 99%

ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

Lin¹,

Yang²,

Yang

et al. 2005

Molecular and Cellular Biology

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We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5 splice site (5ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5ss, while the mutants still interacted with the U1-5ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.mRNA precursors (pre-mRNA) are spliced in a large RNAprotein complex, the spliceosome (12,30,42). Spliceosome assembly requires precise recognition of each of the splice sites, namely, the 5Ј splice site (5Јss), branch point, and 3Ј splice site. 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 5Јss by the U1 snRNP, along with binding of the polypyrimidine tract and the 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 (1,14,29). After an extensive rearrangement, a two-step chemical reaction is catalyzed by the spliceosome to remove the intron from the pre-mRNA.Pre-mRNA splicing is remarkably accurate. The splicing accuracy is achieved by inspection of the individual splice site multiple times by multiple factors (33). Recognition of the 5Ј splice site is an early event in the pre-mRNA splicing process. The 5Јss is recognized by 5-to 7-base-pair interactions between the 5Јss and 5Ј end of the U1 snRNA (44). Recent studies suggest that prior to the recognition of the 5Јss by the U1-5Јss RNA-RNA base pair interactions the 5Јss is recognized by a protein factor, the U1 snRNP U1C. It is believed that binding of U1C to the 5Јss helps the base pair interactions between U1 and the 5Јss (3, 48). The U1-5Јss duplex is unwound to expose the same 5Јss sequence for pairing with the U6 snRNA prior to the first-step chemical re...

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Section: Discussionmentioning

confidence: 99%

ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

Lin¹,

Yang²,

Yang

et al. 2005

Molecular and Cellular Biology

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“…Plasmids expressing WT and K53R/E55A mutant p68 included untagged versions in pcDNA3 (Invitrogen), as well as myc epitope-tagged versions in pSG5 (Stratagene, La Jolla, CA, USA), GAL4-tagged versions (in a pcDNA3 derivative expressing amino acids 1-147 of GAL4 (Wilson et al, 2004)) and GST-tagged versions in pEBG2T (Ogilvie et al, 2003). Plasmids expressing His-tagged SUMO-1, SUMO-2 and SUMO-3 were generated in pcDNA3; the plasmid expressing myc-tagged PIAS1 was in pSG5; plasmids expressing HAtagged PIAS1 and PIASy were in pKW2T (a gift from Mienrad Busslinger, Vienna Biocentre, Austria); Luciferase reporter plasmids included the p53-responsive PG13-Luc (el-Deiry et al, 1993) and pTK-Luc bearing two copies of the GAL4-binding site (a gift from Brian Wilson, McGill University, Montreal, Canada).…”

Section: Plasmidsmentioning

confidence: 99%

“…GST pulldowns GST-tagged p68 and vector control were expressed in 293 cells as described previously (Ogilvie et al, 2003) and purified on glutathione beads using standard conditions. PIAS1 and PIASy were translated in vitro using the TNT kit from Promega and GST pulldowns were performed as described previously (Hsieh et al, 1999).…”

Section: Antibodiesmentioning

confidence: 99%

SUMO modification of the DEAD box protein p68 modulates its transcriptional activity and promotes its interaction with HDAC1

et al. 2007

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The nuclear protein p68 (also known as Ddx5) is a prototypic member of the 'DEAD box' family of RNA helicases, which has been shown to be abnormally expressed and modified in colorectal tumors and to function as an important transcriptional regulator. Here, we show that p68 is modified in vivo on a single site (K53) by the small ubiquitin-like modifier-2 (SUMO-2). We demonstrate that the SUMO E3 ligase PIAS1 interacts with p68 and enhances its SUMO modification in vivo. To determine the functional consequences of SUMO modification, we compared the transcriptional activity of p68 and a K53R mutant that could not be SUMO-modified. Our data show that SUMO modification enhances p68 transcriptional repression activity and inhibits the ability of p68 to function as a coactivator of p53. These findings may be explained by the ability of wild type, but not K53R p68, to alter the modification state of chromatin by recruitment of histone deacetylase 1 (HDAC1).

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“…P72, a human nuclear DEAD box protein and a very close homologue of P68, has recently been shown to form heterodimers with P68 in vivo (Ogilvie et al, 2003). P72 has a predominantly granular nucleoplasmic staining pattern, excluding nucleoli, like P68 (Lamm et al, 1996).…”

Section: Intranuclear Localisation Of P68mentioning

confidence: 99%

Satellite DNA binding and cellular localisation of RNA helicase P68

Enukashvily

Donev

Sheer

et al. 2005

Journal of Cell Science

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We purified a 68-kDa protein from the mouse nuclear matrix using ion exchange and affinity chromatography. Column fractions were tested for specific binding to mouse minor satellite DNA using a gel mobility shift assay. The protein was identified by mass spectrometry as RNA helicase P68. In fixed cells, P68 was found to shuttle in and out of SC35 domains, forming fibres and granules in a cell-cycle dependent manner. Analysis of the P68 sequence revealed a short potential coiled-coil domain that might be involved in the formation of P68 fibres. Contacts between centromeres and P68 granules were observed during all phases of the cycle but they were most prominent in mitosis. At this stage, P68 was found in both the centromeric regions and the connections between chromosomes. Direct interaction of P68/DEAD box RNA helicase with satellite DNAs in vitro has not been demonstrated for any other members of the RNA helicase family.

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The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells

Cited by 78 publications

References 38 publications

ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

SUMO modification of the DEAD box protein p68 modulates its transcriptional activity and promotes its interaction with HDAC1

Satellite DNA binding and cellular localisation of RNA helicase P68

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