Characterization of interactions among the Cef1p-Prp19p-associated splicing complex

Ohi, Melanie D.; Gould, Kathleen L.

doi:10.1017/s1355838202025050

Cited by 104 publications

(156 citation statements)

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“…Cwc2 also comigrated with Prp19 and other core NTC proteins in the glycerol gradient (Table 2). However, it is only loosely associated with the NTC complex (Ohi and Gould 2002), which may explain the low precipitation efficiency of the U2 snRNP and IL RNA. Taken together, our immunoprecipitation results indicate that a part of the NTC complex remains associated with a fraction of the U2 and U5 snRNPs as well as the IL RNA after ILS dissociation by the NTR complex.…”

Section: Cold Spring Harbor Laboratory Press On March 24 2019 -Publimentioning

confidence: 99%

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

et al. 2013

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The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20-25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the ''nineteen complex'' associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.[Keywords: intron-lariat spliceosome; disassembly; Prp22; Prp43; Brr2] Supplemental material is available for this article. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous nonsnRNP proteins (Will and Lü hrmann 2011). The snRNPs are involved in recognizing short conserved sequences of the pre-mRNA, including the 59 and 39 splice sites (SS) and the branch site (BS), and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. These events are mediated by at least eight conserved DExD/H-box NTPases (hereafter termed RNA helicases) that act at discrete stages of the splicing pathway (Staley and Guthrie 1998;Cordin et al. 2012).Spliceosome assembly is initiated by the binding of U1 and U2 snRNPs to the 59 and the BS, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP, forming the precatalytic B complex. Catalytic activation of the spliceosome (leading to complex B act ) involves the displacement of U1 and U4 snRNAs from the spliceosome and the formation of new base pair interactions between the U6 and U2 snRNAs and the 59 SS (Staley and Guthrie 1998). The resulting RNA structure plays a central role in catalyzing both steps of splicing (Nilsen 1998).These RNA-RNA rearrangements are remodeled by the combined actions of the RNA helicases Prp28 and Brr2, which disrupt the base-pairing between U1 snRNA and the 59 SS and between the U4 and U6 snRNAs, respectively (Laggerbauer et al. 1998;Raghunathan and Guthrie 1998;Staley...

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Section: Cold Spring Harbor Laboratory Press On March 24 2019 -Publimentioning

confidence: 99%

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

et al. 2013

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Section: Introductionmentioning

confidence: 99%

“…Based on epistasis analysis PSO4 was assigned to both the yeast RAD6 and RAD52 groups indicating the pleiotropic nature of this mutation [3,4]. Interestingly, cloning of PSO4 showed that it was allelic to PRP19 a previously characterized component of the pre-mRNA splicing complex in both yeast and human cells [5][6][7][8][9][10].The complete function of Prp19 in mRNA splicing is not known, however, it has been shown that the Prp19p-associated complex is required for activation of the pre-mRNA splicesome and for the stable association of the small nuclear RNAs U5 and U6 with the spliceosome after U4 is dissociated [11,12]. These findings suggest a possible structural role for the Prp19-associated complex in pre-mRNA splicing.…”

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confidence: 99%

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

Legerski²

2007

Biochemical and Biophysical Research Communications

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Prp19/Pso4, a U-box containing E3 ligase, has a demonstrated role in pre-mRNA splicing, but has also been implicated in both yeast and mammalian cells as having a direct role in DNA damage processing. In this report we provide further evidence in support of this latter assertion. We show that hPrp19 forms an ubiquitylated oligomeric species that is resistant to disruption by SDS gel electrophoresis under nonreducing conditions suggesting that is mediated by a thiolester between ubiquitin and a cysteine residue in Prp19. The level of this species is significantly enhanced upon treatment of cells with DNA damaging agents, and its association with chromatin is increased. In addition, hPrp19 is known to form a stable core complex with Cdc5L, Plrg1, and Spf27; however, ubiquitylated hPrp19 fails to interact with either Cdc5L or Plrg1 indicating that DNA damage can induce profound alterations to the hPrp19 core complex. Finally, we show that overexpression of hPrp19 in human cells provides a pro-survival affect in that it reduces the levels of apoptosis observed after exposure of cells to DNA damage.The pso4-1 mutant of S. cerevisiae was isolated in a genetic screen for strains sensitive to the cross-linking agent psoralen plus UVA (PUVA) [1,2]. Characterization of this mutant indicated that it was particularly sensitive to bifunctional alkylating agents, but was also sensitive to a broad range of DNA damaging agents including IR, UV, and monofuntional alkylating compounds. Induced mutagenesis and induced and spontaneous mitotic recombination were all greatly reduced in this mutant suggesting that Pso4 is involved in a recombinational pathway of error-prone repair. Based on epistasis analysis PSO4 was assigned to both the yeast RAD6 and RAD52 groups indicating the pleiotropic nature of this mutation [3,4]. Interestingly, cloning of PSO4 showed that it was allelic to PRP19 a previously characterized component of the pre-mRNA splicing complex in both yeast and human cells [5][6][7][8][9][10].The complete function of Prp19 in mRNA splicing is not known, however, it has been shown that the Prp19p-associated complex is required for activation of the pre-mRNA splicesome and for the stable association of the small nuclear RNAs U5 and U6 with the spliceosome after U4 is dissociated [11,12]. These findings suggest a possible structural role for the Prp19-associated complex in pre-mRNA splicing. Mammalian Prp19 associates with a large number of splicing factors, although, it appears to form a core complex with three other proteins including Cdc5L, Plrg1, and Spf27 [9]. The structure of the Prp19 core complex is not completely resolved, however, it has been shown that yeast Prp19 forms a tetramer via its coiled coil domains, and that the tetramer interacts with one copy of Cdc5L through the associated coiled coil domains *Corresponding author. Fax. 713-792-1474; E-Mail: rlegersk@mdanderson.org. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

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“…In recent years, several complexes have been identified that seem to challenge previous models of spliceosome formation. These include the yeast Cwf/Cwc complex, 7,8 lnRNP complexes, 9 a complex larger than 150S with base paired U2 and U6, 10 a U2/U4/U5/U6-containing pseudospliceosome complex, 11 a pentasnRNP from S. cerevisiae, 12 and a HeLa nuclear extract penta-snRNP complex. 13 The exact functional relevance of these complexes remains to be determined, none have been shown to be capable of splicing on their own.…”

Section: Introductionmentioning

confidence: 99%

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

Peng¹,

Hawkins²,

Link³

et al. 2006

RNA Biology

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Previously published online as a RNA Biology E-publication: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=3017 KEY WORDS Research PaperThe Splicing Factor PSF is Part of a Large Complex that Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs ABSTRACT PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4˚C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.

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Characterization of interactions among the Cef1p-Prp19p-associated splicing complex

Abstract: Schizosaccharomyces pombe (Sp) Cdc5p and its Saccharomyces cerevisiae (Sc)

Cited by 104 publications

References 65 publications

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

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