The human Ski-interacting protein functionally substitutes for the yeast PRP45 gene

doi:10.1016/j.bbrc.2004.05.096

Biochemical and Biophysical Research Communications

Volume 319, Issue 4, 9 July 2004, Pages 1105-1109

https://doi.org/10.1016/j.bbrc.2004.05.096 Get rights and content

Abstract

The PRP45 gene has been identified as encoding a protein involved in mRNA splicing that is essential in Saccharomyces cerevisiae. PRP45’s human homolog SKIP has been identified as a mediator of transcriptional programming in a variety of signal transduction pathways including TGFβ, nuclear hormones, Notch, and retinoblastoma signaling. However, Skip has been also identified in purified spliceosomal complexes but an explicit role in splicing has not been identified in mammalian cells. To determine if the Skip protein could function as a splicing factor we investigated if the SKIP gene could functionally complement the yeast PRP45 gene. We show that SKIP complements the PRP45 deletion and rescues the lethal phenotype. These results show that the human SKIP gene can functionally substitute for the mRNA splicing gene PRP45 of S. cerevisiae.

Section snippets

Materials and methods

Yeast strain and media. The yeast strain used was obtained from Research Genetics the genotype is MATa/αhis3Δ1/his3Δ1 leu2Δ/leu2Δ lys2Δ/LYS2 MET15/met15Δura3Δ/ura3Δ (4741/4742) PRP45/prp45Δ kanmx4. Media were prepared as described in [19].

Plasmid construction. The human Ski-interacting protein was cloned from pCS2Skip and digested with BamHI and XbaI, where the XbaI site was blunted. The insert was cloned into the yeast shuttle vector p426ADH [20] plasmid using a 5^′BamHI and blunted 3^′EcoRI

Amino acid alignment of Skip, Bx42, and Prp45 shows two regions of homology

Amino acid alignment of the polypeptide sequences of Skip from Homo sapiens (AAC15912.1), Bx42 from Drosophila melanogaster (P39736), and Prp45 from S. cerevisiae (NP_009370.1) is shown in Fig. 1. As can be seen there is a much greater degree of homology between the human and Drosophila proteins than there is with Prp45. This extensive homology is also seen with the Skip homologs from mouse, S. pombe and all the other Skip homologs described to date (data not shown). In marked contrast, Fig. 1

Acknowledgements

We thank Rolf Sternglanz for his help in writing the manuscript and the Sternglanz lab for providing reagents and technical assistance; Jean Beggs and Michael Albers for initial work that spurred these studies; K. Donnelly for supporting our research project; and all the members of our laboratory for helpful discussions and criticism. This work was supported by Grant CA42573 from the National Institutes of Health to M.J.H.

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Citation Excerpt :
Prp45 is the SKIP homolog in yeast (Gahura et al., 2009). Weak prp45 alleles exhibit a temperature-sensitive growth phenotype, while strong prp45 alleles are lethal (Figueroa and Hayman, 2004; Gahura et al., 2009). Human SKIP is necessary for the expression of p21, and defects in SKIP result in subsequent p53-mediated apoptosis (Chen et al., 2011).
Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5′ and 3′ splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.
SKIP and BIR-1/Survivin have potential to integrate proteome status with gene expression
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SKIP also directly binds the retinoblastoma tumor suppressor protein pRb and, in co-operation with Ski, overcomes the G1 arrest induced by pRb [45]. SKIP is also involved in regulation of splicing [12,13,46,47]. Thus, SKIP has a well-documented role in the regulation of transcription and cell cycle.
SKIP and BIR are evolutionarily conserved proteins; SKIP (SKP-1) is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Their loss of function induces overlapping developmental phenotypes. We searched for SKP-1 and BIR-1 interaction on protein level using yeast two-hybrid screens and identified partially overlapping categories of proteins as SKIP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement in multiple protein complexes. To visualize the effect of BIR-1 on the proteome in Caenorhabditis elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS3 and RPL5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated protein. Together, these results suggest that SKP-1 and BIR-1 are multifunctional proteins that form multiple protein complexes in both shared and distinct pathways and have the potential to connect proteome signals with the regulation of gene expression.
The genomic organization of the genes encoding BIR-1 and SKIP (SKP-1) in C. elegans have suggested that these two factors, each evolutionarily conserved, have related functions. However, these functional connections have remained elusive and underappreciated in light of limited information from C. elegans and other biological systems. Our results provide further evidence for a functional link between these two factors and suggest they may transmit proteome signals towards the regulation of gene expression.
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Skip binds to the Ski oncogene to synergistically overcome RB-mediated transcriptional repression and cell cycle arrest [3]. Skip is the functional orthologue of the yeast splicing factor Prp45 and thus physically links RB to RNA splicing control [4]. RB also physically binds to Pur-alpha, a protein with the ability to bind both single-stranded DNA and RNA.
The retinoblastoma tumor suppressor protein (RB) is inactivated in a majority of cancers. RB restricts cell proliferation by inhibiting the E2F family of transcription factors. The current model for RB/E2F function describes its role in regulating transcription at gene promoters. Whether the RB or E2F proteins might play a role in gene expression beyond transcription initiation is not well known. This review describes evidence that points to a novel role for the RB/E2F network in the regulation of RNA processing, and we propose a model as a framework for future research. The elucidation of a novel role of RB in RNA processing will have a profound impact on our understanding of the role of this tumor suppressor family in cell and developmental biology.
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This chapter focuses on the regulation and the influence of splicing on two important cellular processes: programmed cell death (apoptosis) and cell cycle control. Apoptosis is a process that removes deleterious or useless cells during animal development and is one of many cellular processes in which alternative splicing plays an important regulatory role. Oligonucleosomal DNA fragmentation is one of the hallmarks of apoptotic cell death. The cell cycle is a collection of highly ordered processes that lead to the duplication of a cell. Specific splicing factors are required for cell cycle progression by modulating splicing of transcripts encoding cell cycle regulators. In metazoan organisms, most transcripts synthesized by RNA polymerase (RNAP) II contain non-coding intervening sequences called introns, which must be accurately and efficiently removed by the process of pre-mRNA splicing to form translatable mRNAs. Alternative splicing is the removal of introns in different combinations. It produces diverse mature mRNAs encoding structurally and functionally distinct protein isoforms from a single gene. Alternative splicing is widely involved in gene expression in higher eukaryotes, especially in vertebrates. Pre-mRNA splicing takes place within a large molecular complex, the spliceosome, that is composed of five small nuclear RNA and a large number of protein factors. Regulation of alternative splicing largely relies on a broad spectrum of interactions between sequence elements in the mRNA precursor and a complex repertoire of protein factors. Shifting the balance between alternatively spliced isoforms of a given pre-mRNA is important in modulating both programmed cell death (apoptosis) and cell cycle control.
Reprogramming nucleolar size by genetic perturbation of the extranuclear Rab GTPases Ypt6 and Ypt32
2024, FEBS Letters
Cloning and functional analysis of BdSKIP gene from Brachypodium distachyon
2023, Zhiwu Shengli Xuebao/Plant Physiology Journal

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Amino acid alignment of Skip, Bx42, and Prp45 shows two regions of homology

Acknowledgements

References (21)

Integrating mRNA processing with transcription

Cell

A unified theory of gene expression

Cell

Ski-interacting protein interacts with Smad proteins to augment transforming growth factor-beta-dependent transcription

J. Biol. Chem.

Ternary complexes and cooperative interplay between NCoA-62/Ski-interacting protein and steroid receptor coactivators in vitamin D receptor-mediated transcription

J. Biol. Chem.

NCoA62/SKIP is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing

J. Biol. Chem.

The homolog of chromatin binding protein Bx42 identified in Dictyostelium

Gene

The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity

Mech. Dev.

Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds

Gene

Ski-interacting protein, a bifunctional nuclear receptor coregulator that interacts with N-CoR/SMRT and p300

Biochem. Biophys. Res. Commun.

Alternative pre-mRNA splicing and proteome expansion in metazoans

Nature

Cited by (19)

SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis

SKIP and BIR-1/Survivin have potential to integrate proteome status with gene expression

The RB/E2F pathway and regulation of RNA processing

The Role of Alternative Splicing During the Cell Cycle and Programmed Cell Death

Reprogramming nucleolar size by genetic perturbation of the extranuclear Rab GTPases Ypt6 and Ypt32

Cloning and functional analysis of BdSKIP gene from Brachypodium distachyon