Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae

Russell, Caroline S.; Ben-Yehuda, Sigal; Dix, Ian; Kupiec, Martin; Beggs, Jean D.

doi:10.1017/s1355838200000984

Cited by 57 publications

(53 citation statements)

References 40 publications

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“…Other groups have previously noted numerous twohybrid interactions between Cef1p, Syf1p, Clf1p, Ntc30p/ Isy1p, andNtc20p (Ben-Yehuda et al+, 1998, 2000b;Dix et al+, 1998;Russell et al+, 2000), although it has not been appreciated that these interactions occur within the context of a stable complex+ Although we have not yet placed Ntc30p/Isy1p or Ntc20p within the complex, we have made significant progress in mapping protein interactions between identified and previously unidentified Ntc components+ Interestingly, we have been unable to detect an interaction between Ntc30p/Isy1p and Cef1p that was reported by Ben-Yehuda et al+ (2000b)+ Such an interaction was also not detected by far western analysis (Tsai et al+, 1999;Chen et al+, 2001)+ While our paper was under revision, complementary conclusions regarding the identity of Ntc77p as Clf1p and Ntc90p as Sylf1p were published by Chen et al+ (2002)+ These authors also determined that Clf1p and Syf1p interacted with Cef1p, Ntc20p, and Isy1p (Ntc30p) by two-hybrid analysis+ Of significance to understanding Cdc5p/Cef1p function was our finding that a mutation in PRP19 led to loss of Cef1p+ This indicates that Cef1p/Cdc5p function depends on its interaction with Prp19p+ In light of this finding, although a number of reports have suggested a role for hCDC5 in transcriptional regulation (Hirayama & Shinozaki, 1996;Bernstein & Coughlin, 1997Groenen et al+, 1998), it is likely that Cdc5p/Cef1p is involved primarily in pre-mRNA processing, as no transcriptional function has been attributed to Prp19p+…”

Section: Discussionmentioning

confidence: 99%

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

Ohi

Gould

2002

RNA

104

146

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Section: Discussionmentioning

confidence: 99%

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

Ohi

Gould

2002

RNA

104

146

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“…Clf1-HA). The bulky TAP epitope enhances snRNA recovery, since previous experiments performed with alternative CLF1 alleles failed to recover snRNA (10) or recovered no U2 snRNA and nearly background levels of U5 and U6 snRNA (15). The amount of snRNA recovered with Clf1-TAP is less than what is commonly observed with snRNP-specific proteins, however.…”

Section: Clf1pmentioning

confidence: 93%

“…When these gradient fractions are used for immune precipitation, the majority of Clf1-TAP-associated snRNA is recovered from the poly-snRNP fraction (fractions [15][16][17][18][19] with no detectable free U6 snRNP (Fig. 2D, fractions 5-7; see Fig.…”

Section: Clf1pmentioning

confidence: 99%

“…In humans, the crooked neck protein is a stable component of the spliceosome, recruited at or near the time of U4/U6.U5 tri-snRNP addition (14). In yeast, genetic and biochemical studies implicate Clf1p in a number of steps in spliceosome assembly and in both catalytic steps in splicing (10,(15)(16)(17)(18). This broad spectrum of Clf1p interactions is consistent with the 15 TPR repeats of Clf1p serving as a platform for the recruitment of splicing factors during spliceosome assembly.…”

mentioning

confidence: 99%

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The Clf1p Splicing Factor Promotes Spliceosome Assembly through N-terminal Tetratricopeptide Repeat Contacts

Wang

Hobbs

Lynn

et al. 2003

Journal of Biological Chemistry

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Spliceosome assembly follows a well conserved pathway of subunit addition that includes both small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splicing factors. Clf1p is an unusual splicing factor composed almost entirely of direct repeats of the tetratricopeptide repeat (TPR) protein-binding motif. Here we show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex (nineteen complex; NTC) and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs. Thirty Ccf (Clf1p complex factor) proteins have been identified by mass spectroscopy or immune detection as known or suspected components of the yeast spliceosome. Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p protein (i.e. Clf1⌬2-TAP) destabilizes Clf1p complexes assembled in vivo, causing the release of the Cef1p and Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and Hsh155p snRNP proteins. In vitro, temperature inactivation of Clf1⌬2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex. These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.The spliceosome is composed of five small nuclear RNAs (snRNAs) 1 and over 70 distinct polypeptides (reviewed in Refs. 1 and 2; see references in Refs. 3 and 4). Numerous studies have demonstrated that in vitro spliceosome assembly progresses through an ordered sequence that is largely conserved from yeast to humans. By tracking assembly through small nuclear ribonucleoprotein (snRNP) association with pre-mRNA, it has been shown that the U1 snRNP binds independently other snRNP particles in an ATP-independent step said to commit the pre-mRNA to the splicing pathway. U2 snRNP is recruited next in an ATP-dependent step that forms the prespliceosome. Finally, the U4, U5, and U6 snRNAs are jointly recruited as the exceptionally large U4/U6.U5 trisnRNP particle to produce the spliceosome. Together, the U1/U2 snRNP-dependent prespliceosome and the U4/U6.U5 tri-snRNP particle account for all of the snRNAs and the majority of proteins known to act in splicing. A more limited number of non-snRNP splicing factors bind directly to the pre-mRNA substrate to promote snRNP addition or promote conformational changes within the splicing complex following snRNP addition (1-3).Regulated pre-mRNA splicing events are driven by proteins that act to promote or block the recruitment of constitutive splicing factors (4, 5). The major snRNP addition steps are clear targets of this regulation. Whereas comparatively few pre-mRNAs appear regulated in yeast, this system does offer a valuable means to characterize the basic pathway for splicing factor association and hence to identify critical steps in assembly and possible targets for regulation. The addition of the U1 and U2 snRNP particles to the spl...

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“…17 Different snRNPs and accessory proteins are recruited or excluded at different steps of the splicing process, resulting in a very dynamic composition of the spliceosome machine and the generation of extensive rearrangements during different stages of splicing. 17,18 A connection between the spliceosome and cell cycle progression has been found in many organisms including budding yeast, [19][20][21][22][23][24] fission yeast, [25][26][27] Drosophila, 9,28 chicken, 29 mouse, 30 and human cells. 6,11,12,29,31,32 In human cells, depletion of different spliceosome components with siRNAs results in multiple cell cycle defects, with most siRNAs analyzed eliciting mitotic defects 6,11,12,31 although accumulation of cells in S phase 32 has also been observed.…”

Section: Introductionmentioning

confidence: 99%

Graded requirement for the spliceosome in cell cycle progression

Karamysheva¹,

Díaz-Martínez

Warrington

et al. 2015

Cell Cycle

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Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions.

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Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae

Cited by 57 publications

References 40 publications

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

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

The Clf1p Splicing Factor Promotes Spliceosome Assembly through N-terminal Tetratricopeptide Repeat Contacts

Graded requirement for the spliceosome in cell cycle progression

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