A three-hybrid system to detect RNA-protein interactions in vivo.

SenGupta, Dhruba J.; Zhang, Beilin; Kraemer, Brian C.; Pochart, Pascale; Fields, Stanley; Wickens, Marvin

doi:10.1073/pnas.93.16.8496

Cited by 478 publications

(450 citation statements)

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“…Mary Wickens, Stan Fields, and coworkers have recently devised a three-hybrid screen, a variation of the yeast two-hybrid technology, for selecting RNA-binding proteins in yeast (SenGupta et al 1996). Briefly, a vector expressing the lexA DNA-binding domain fused with the phage MS-2 protein was integrated into the yeast genome.…”

Section: Cloning Of the Slbpmentioning

confidence: 99%

“…A diagram of the strategy, adapted from Wickens and coworkers (SenGupta et al 1996), is shown in Figure 1A. Two genes, his3 conferring resistance to aminotriazole and lacZ encoding f~-galactosidase, which will turn the colonies blue in the presence of X-Gal, will be activated by the Gal4 activation domain fused with a eDNA that binds specifically to the RNA "bait."…”

Section: Cloning Of the Slbpmentioning

confidence: 99%

“…(A) The yeast three-hybrid system. A diagram of the strategy for the yeast threehybrid system adapted from Wickens and co-workers is shown (SenGupta et al 1996). The lexA-MS-2 fusion protein binds to the MS2 RNA-binding site in the RNA and the Gal4-cDNA fusion binds to the target RNA resulting in activation of both the lacZ and his3 gene in the yeast.…”

Section: Cloning Of the Slbpmentioning

confidence: 99%

“…Thus, the SLBP is an example of a protein that may shuttle between the nucleus and the cytoplasm. Using a yeast three-hybrid selection system for RNA-binding proteins (SenGupta et al 1996), we have cloned the cDNA for the SLBP from humans and frogs. The mammalian SLBP is a 31-kD protein that is not related to other proteins in the data base and that contains a novel 73-amino-acid RNA-binding domain.…”

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confidence: 99%

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The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

Wang¹,

Whitfield²,

Ingledue³

et al. 1996

Genes Dev.

249

262

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Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.

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Section: Cloning Of the Slbpmentioning

confidence: 99%

Section: Cloning Of the Slbpmentioning

confidence: 99%

Section: Cloning Of the Slbpmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

Wang¹,

Whitfield²,

Ingledue³

et al. 1996

Genes Dev.

249

262

View full text Add to dashboard Cite

show abstract

“…(B) Plate assays to test the contribution to RNA binding of the [C157S, C163S, H167I] small subunit ZBDII mutant in the modified RNA three-hybrid system (Webb and Wise, 2004). The strength of the RNA-protein interaction is reflected by growth in the presence of increasing amounts of 3-amino-triazole (SenGupta et al, 1996; Eckman et al, 2002). The data shown are for the human ␤-globin 3Ј splice site that is similar to many S. pombe 3Ј splice sites; its sequence extending from just downstream of the branchpoint to 10 bases beyond the 3Ј AG dinucleotide is shown beneath the plate assays.…”

mentioning

confidence: 99%

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

Webb

Lakhe-Reddy

Romfo

et al. 2005

MBoC

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The heterodimeric splicing factor U2AF plays an important role in 3 splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3 splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AF LG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3 splice site recognition, we sought introns for which the spU2AF LG and spU2AF SM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3 pyrimidine tract. These and other studies performed in fission yeast support a model for 3 splice site recognition in which the two subunits of U2AF functionally collaborate in vivo. INTRODUCTIONThe removal of noncoding introns and the joining of coding exons via splicing, an essential step in the eukaryotic premRNA processing pathway, requires accurate splice site selection by the spliceosome. Initial recognition of the 5Ј exon/intron boundary is achieved via base pairing with the U1 snRNA component of the U1 snRNP (Zhuang and Weiner, 1986), whereas U2AF (U2 snRNP auxiliary factor) is the first factor bound to the 3Ј splice site (Ruskin et al., 1988). Biochemical complementation assays demonstrated that U2AF is required for the subsequent ATP-dependent association of U2 snRNP with pre-mRNA branchpoints (Ruskin et al., 1988;Valcarcel et al., 1996). Purified U2AF is a heterodimer composed of large and small subunits in humans (Zamore and Green, 1989), Drosophila melanogaster (Kanaar et al., 1993;Rudner et al., 1996), Caenorhabditis elegans (Zorio et al., 1997; Zorio and Blumenthal, 1999) and Schizosaccharomyces pombe (Potashkin et al., 1993;Wentz-Hunter and Potashkin, 1996). Functional conservation of this splicing factor is evidenced by 1) the restoration of splicing activity to U2AF-depleted HeLa nuclear splicing extracts via addition of Drosophila U2AF large subunit (dmU2AF LG ; Zamore and Green, 1991) and 2) the ability of human U2AF 35 (hsU2AF SM ) to restore growth to an S. pombe strain lacking the small subunit (Webb and Wise, 2004).The structural domains of U2AF are also conserved except in Saccharomyces cerevisiae, where the large subunit is highly divergent and the small subunit is absent entirely (Abovich et al., 1994). The small subunit of U2AF consists of two zinc-binding domains (ZBDs) surrounding a central pseudo-RNA recognition motif (⌿RRM; Rudner et al., 1998b), also known as a PUMP (PUF60/U2AF/M...

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Unraveling the role of helicases in transcription

Eisen

Lucchesi

1998

Bioessays

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A three-hybrid system to detect RNA-protein interactions in vivo.

Abstract: RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in

Cited by 478 publications

References 40 publications

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

Unraveling the role of helicases in transcription

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