Interactions between PRP9 and SPP91 splicing factors identify a protein complex required in prespliceosome assembly.

Legrain, Pierre; Chapon, C; Galisson, Frédérique

doi:10.1101/gad.7.7b.1390

Cited by 50 publications

(47 citation statements)

References 52 publications

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“…Interaction as a unit is supported by evidence that PRP5, PRP9, PRPJ1, and PRP21 show synthetic lethal interactions with one another (46), and a suppressor of prp9-1 called SPP91 (11) is an allele of PRP21 (2). Results from the two-hybrid system have also detected protein-protein interactions between Prp2lp and both Prp9p and Prpllp (30,31 Fig. 1C and 2).…”

Section: Methodssupporting

confidence: 54%

“…2) do not distinguish between models involving sequential or simultaneous interaction of the proteins with U2. Although genetic data (11,46) and results from the two-hybrid system (30,31) suggest that proteins of this group interact, we wanted to determine if the three proteins are in the same complex at the same time with pre-mRNA. We performed a double-immunoprecipitation experiment (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Genetic suppression data (11) and results using the yeast two-hybrid system for detecting protein-protein interactions (14,18) show that PRP21 interacts with PRP9 (30,31). Similarly PRPII interacts with PRP21 and possibly indirectly with PRP9 through its interaction with PRP21 (30).…”

mentioning

confidence: 99%

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Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Wells¹,

Ares²

1994

Mol. Cell. Biol.

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Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10prp mutations and found lethal double-mutant combinations withprpS, -9, -11, and -21 but not with prp3, 4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prpllp, and Prp2lp) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -llp, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop Ila and mediate the association of the U2 snRNP with pre-mRNA.Splicing of nuclear pre-mRNA requires a sophisticated ribonucleoprotein (RNP) complex called the spliceosome. The spliceosome is built on an intron-containing transcript by the sequential binding of small nuclear RNPs (snRNPs) to each other and to specific sites on the transcript, so that the pre-mRNA is properly arranged for splicing (20,21). Before, during, and after the cleavage-ligation steps of splicing take place, the spliceosome is acted upon by a series of extrinsic factors, some of which bind transiently and trip a limited set of steps in the sequence (15,29,48). The events that define the transition from one state to the next during spliceosome assembly and function are both compositional and conformational: snRNPs and extrinsic factors are added and removed, and RNA-RNA interactions between the small nuclear RNAs (snRNAs) or between the snRNAs and the pre-mRNA within the spliceosome may be established and dissolved (42). Splicing factors include a family of proteins with ATP binding domains suggesting a means for driving the process forward and ensuring the accuracy of events (20,21).The expectation that alternative splicing is achieved by regulated spliceosome assembly has placed the early steps of splicing complex formation under close scrutiny. Using substrate commitment experiments, several investigations have led to a similar set of conclusions for both systems (27,32,38,49,50

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Section: Methodssupporting

confidence: 54%

Section: Methodsmentioning

confidence: 99%

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Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Wells¹,

Ares²

1994

Mol. Cell. Biol.

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“…The monoclonal mouse antibodies used were directed against the U1-70K (HI1) (22) URA::GALJ-lacZ) was transformed with the two-plasmids and transformants were selected on minimal medium lacking leucine and tryptophan and assayed for 0-galactosidase activity using a filter assay with X-gal. Several colonies were streaked and cells were grown in liquid cultures to perform quantitative P-galactosidase assays (13).…”

Section: Immunodetection Of Ul Snrnp Proteins On Western Blotmentioning

confidence: 99%

“…Neither one of the proteins U1C, PRP6, PRP9 or PRP1 1, however, has actually been shown to bind zinc. Recently, Legrain and co-workers (13) demonstrated in the yeast two hybrid system that the two CH motifs in the PRP9 protein are required for the formation of PRP9 homodimers.…”

Section: Introductionmentioning

confidence: 99%

Homodimerization of the human U1 snRNP-specific protein C

Gunnewiek¹,

Aarssen²,

Wassenaar³

et al. 1995

Nucl Acids Res

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The Ul snRNP-specific protein C contains an N-terminal zinc finger-ike CH motif which is required for the binding of the U1C protein to the Ul snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study we demonstrate that the human UIC protein is able to form homodimers as well. Ul C homodimers are found when (i) the human U1C protein is expressed in Escherichia coil, (ii) immunoprecipitations with anti-UI C antibodies are performed on in vitro translated Ul C, and when (iii) the yeast two hybrid system is used. Analyses of mutant Ul C proteins in an in vitro dimerization assay and the yeast two hybrid system revealed that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization.

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