cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity

Keck, T; Glaser, Tova; Rothbarth, Karsten; Stammer, Hermann; Werner, Dieter; Spieß, Eberhard; Nehls, Peter; Greferath, Ruth

doi:10.1093/nar/26.5.1160

Cited by 28 publications

(41 citation statements)

References 14 publications

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“…The absence of Rrp47p did not significantly affect either the expression level of Rrp6p or its coimmunoprecipitation with Rrp4p (R. Houalla and D. Tollervey, unpublished observations), suggesting that Rrp47p is not required for Rrp6p expression or its assembly into the exosome. Although Rrp47p shares no similarity with characterized 3Ј35Ј exoribonucleases, the homologous human protein C1D binds strongly to nucleic acids (28). Rrp47p may therefore function in substrate recruitment and targeting to Rrp6p.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

Mitchell

Petfalski

Houalla

et al. 2003

Molecular and Cellular Biology

149

210

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Related exosome complexes of 335 exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg 2؉ -labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 335 exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3 processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-⌬ strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3-extended nuclear pre-mRNAs or the cytoplasmic 335 mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.The eukaryotic 18S, 5.8S, and 25S rRNAs (yeast nomenclature is given) are generated from a single large RNA polymerase I transcript by a series of endonucleolytic and exonucleolytic RNA processing reactions (reviewed in reference 39). The earliest detectable transcript in yeast, the 35S precursor rRNA (pre-rRNA), also undergoes extensive posttranscriptional modification involving predominantly pseudouridine formation and ribosyl-2Ј-O-methylation. These modifications are directed to specific nucleotides within the ϳ7-kb-long 35S prerRNA via complementary base-pairing mechanisms involving ϳ70 different small nucleolar RNAs (snoRNAs).The snoRNAs can be divided into two major functional groups; the box C/D snoRNAs direct methylation of ribosyl-2Ј-hydroxyl groups, whereas the H/ACA snoRNAs direct the conversion of uridine to pseudouridine (for reviews, see references 4 and 21). Genes encoding snoRNAs have a varied organization, but in yeast and mammals all are transcribed by RNA polymerase II. Most yeast snoRNA genes are expressed as individual transcripts from their own promoters, whereas several are processed from common primary transcripts and a few are encoded within the introns of protein-coding genes. Gene clusters are the predominant organization of snoRNA genes in plants, whereas the majority of mammalian snoRNAs are intron encoded.The synthesis of the mature 3Ј ends of all characterized snoRNAs requires endonucleolytic cleavage of the transcript, followed by 3Ј35Ј exonucleolytic processing. Endonucleolytic cleavage is by Rnt1p, the yeast RNase III homologue, or by cleavage factor 1A (CF1A), which is also required for the 3Ј end processing of mRNA transcripts (15). Maturation of intron-encoded snoRNAs involves linearization of the excised intron lariat, either by the debranching enzyme Dbr1p or endonucleolytic cleavage, followed by exonucleolytic proces...

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Section: Discussionmentioning

confidence: 99%

“…Rrp47p was previously reported to function in DNA doublestrand break repair and the homologous human protein C1D binds with high affinity to free 3Ј ends of DNA (14,28). Both nonhomologous end joining and homologous recombination events involve nucleotide removal from the free ends by exonucleases.…”

Section: Discussionmentioning

confidence: 99%

Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

Mitchell

Petfalski

Houalla

et al. 2003

Molecular and Cellular Biology

149

210

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“…11,14,64,65 Research on Rrp47 has focussed on its physical association with the exosome nuclease complex. …”

Section: The Exosome Cofactor Rrp47mentioning

confidence: 99%

Rrp6, Rrp47 and Cofactors of the Nuclear Exosome

Butler

Mitchell

2010

Advances in Experimental Medicine and Biology

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This chapter reviews the present state of knowledge on the activity of enzymes that function with the RNA exosome in the nucleus. In this compartment, the exosome interacts physically and functionally with the exoribonuclease Rrp6 and several cofactors, most prominently Rrp47 and the TRAMP complex. These interactions decide the fate of RNA precursors from transcription through the formation of mature ribonucleoprotein particles (RNPs) and the export of the RNPs to the cytoplasm. The nuclear exosome catalyzes the formation of the mature 3' ends of many of these RNAs, but in other cases degrades the RNAs to mononucleotides. Co-factors such as Mpp6, TRAMP and the Nrd1/Nab3 complex play important roles in determining the outcome of the interaction of RNPs with the nuclear exosome. The details that govern the specificity of these decisions remain a rich source for future investigation.4

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“…X95592). C1D is a ubiquitously expressed nuclear protein that has been identified recently by screening a cDNA expression library with monoclonal antibodies raised against the residual polypeptides that remain attached to DNA even following aggressive purification methods that employ SDS, proteinase K, and phenol extraction (Nehls et al 1998; U. Yavuzer and S.P. Jackson, unpubl.; also see Discussion).…”

Section: Identification Of Proteins Interacting With the Putative Lz mentioning

confidence: 99%

“…Although initial experiments were designed to address an interaction between endogenous mammalian cell C1D and DNA-PK cs in coimmunoprecipitation assays, such studies were not feasible because C1D remains tightly bound to DNA during whole cell or nuclear extract preparation and, consequently, is not detectable by Western blotting of soluble cell extracts (Nehls et al 1998; U. Yavuzer, unpubl.). To circumvent this problem, we expressed recombinant C1D in mammalian cells in anticipation that a fraction of the expressed protein would remain soluble following cell extract preparation.…”

Section: Dna-pk Activation Without Dna Ends Genes and Development 2193mentioning

confidence: 99%

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D

Yavuzer¹,

Smith²,

Bliss³

et al. 1998

Genes Dev.

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DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ∼465-kD catalytic subunit, DNA-PK cs . Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PK cs . Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.

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cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity

Cited by 28 publications

References 14 publications

Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

Rrp6, Rrp47 and Cofactors of the Nuclear Exosome

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D

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