Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Chen, Yen-I G.; Moore, Roger E.; Ge, Helen Y.; Young, Mary K.; Lee, Terry D.; Stevens, Scott W.

doi:10.1093/nar/gkm347

Cited by 108 publications

(132 citation statements)

References 100 publications

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“…Cwc23p was first found as a copurifying protein with the Cef1p splicing factor (Ohi et al 2002) and was subsequently identified in other spliceosomal preparations Chen et al 2007). Alternatively reported as essential (Giaever et al 2002) or nonessential (Tizon et al 1999), we find the cwc23TKAN null mutant to be viable but sickly.…”

Section: Discussionmentioning

confidence: 59%

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Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

Pandit

Paul

Zhang³

et al. 2009

Genetics

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Prp43p catalyzes essential steps in pre-mRNA splicing and rRNA biogenesis. In splicing, Spp382p stimulates the Prp43p helicase to dissociate the postcatalytic spliceosome and, in some way, to maintain the integrity of the spliceosome assembly. Here we present a dosage interference assay to identify Spp382p-interacting factors by screening for genes that when overexpressed specifically inhibit the growth of a conditional lethal prp38-1 spliceosome assembly mutant in the spp382-1 suppressor background. Identified, among others, are genes encoding the established splicing factors Prp8p, Prp9p, Prp11p, Prp39p, and Yhc1p and two poorly characterized proteins with possible links to splicing, Sqs1p and Cwc23p. Sqs1p copurifies with Prp43p and is shown to bind Prp43p and Spp382p in the two-hybrid assay. Overexpression of Sqs1p blocks pre-mRNA splicing and inhibits Prp43p-dependent steps in rRNA processing. Increased Prp43p levels buffer Sqs1p cytotoxicity, providing strong evidence that the Prp43p DExD/H-box protein is a target of Sqs1p. Cwc23p is the only known yeast splicing factor with a DnaJ motif characteristic of Hsp40-like chaperones. We show that similar to SPP382, CWC23 activity is critical for efficient pre-mRNA splicing and intron metabolism yet, surprisingly, this activity does not require the canonical DnaJ/Hsp40 motif. These and related data establish the value of this dosage interference assay for finding genes that alter cellular splicing and define Sqs1p and Cwc23p as prospective modulators of Spp382p-stimuated Prp43p function. E IGHT phylogenetically conserved DExD/H-box proteins act at discrete steps to regulate the assembly, activation, and dissociation of the splicing apparatus (reviewed in Brow 2002; Konarska and Query 2005;Linder 2006). How these RNA-dependent ATPases are temporally and functionally regulated remains poorly understood. One putative regulator, the 83-kDa Spp382/Ntr1 protein (henceforth referred to by the Saccharomyces Genome Database standard name, Spp382p), was discovered in a screen for mutants capable of suppressing defects in yeast spliceosome assembly (Pandit et al. 2006). While spp382 null alleles are lethal, partial loss of function suppresses mutations in several other splicing factors, including the genes encoding the essential spliceosomal proteins Prp8p and Prp38p. Spp382p is a spliceosomal protein that binds the Prp43p DExD/H-box protein to promote efficient dissociation of spliceosomal factors after completion of splicing in vitro (Tsai et al. 2005). Some but not all spp382 mutants also accumulate the excised intron product of splicing in vivo, ostensibly due to protection of the intron within a hyperstabilized spliceosome (Pandit et al. 2006;Tanaka et al. 2007). The suppression of spliceosome assembly defects by spp382 mutation is proposed to occur by impairing Spp382p-stimulated dissociation of kinetically impaired or otherwise inefficient spliceosomes by Prp43p (Pandit et al. 2006). Consistent with this hypothesis, prp43 mutations also suppress spliceosome a...

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Section: Discussionmentioning

confidence: 59%

“…At least two DnaJ proteins, DNAJC8 and DNAJC13, copurify with the mammalian spliceosome but neither one has been studied for function in splicing ( Jurica et al 2002;Chen et al 2007). Removal of much of the DnaJ motif from yeast Cwc23p has little obvious consequence on splicing, growth, or this protein's interaction with Spp382p.…”

Section: Discussionmentioning

confidence: 99%

Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

Pandit

Paul

Zhang³

et al. 2009

Genetics

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“…3), together with the previous report that RBM5 stable transformants of lung cancer cell lines undergo apoptosis (29), supports the role of RBM5 as an apoptosis regulator in cancer cells. RBM5 is localized in nuclei and associated with spliceosomal complexes (26,35,36). Our work here indicates that RBM5 binds to casp-2 pre-mRNA and regulates the balance of casp-2L versus casp-2S splicing isoforms, uncovering a previously unknown activity of RBM5 as a splicing regulator.…”

Section: Discussionmentioning

confidence: 61%

Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5

Fushimi

Ray

Kar

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

125

137

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Similar to many genes involved in programmed cell death (PCD), the caspase 2 (casp-2) gene generates both proapoptotic and antiapoptotic isoforms by alternative splicing. Using a yeast RNA-protein interaction assay, we identified RBM5 (also known as LUCA-15) as a protein that binds to casp-2 pre-mRNA. In both transfected cells and in vitro splicing assay, RBM5 enhances the formation of proapoptotic Casp-2L. RBM5 binds to a U/C-rich sequence immediately upstream of the previously identified In100 splicing repressor element. Our mutagenesis experiments demonstrate that RBM5 binding to this intronic sequence regulates the ratio of proapoptotic/antiapoptotic casp-2 splicing isoforms, suggesting that casp-2 splicing regulation by RBM5 may contribute to its tumor suppressor activity. Our work has uncovered a player in casp-2 alternative splicing regulation and revealed a link between the alternative splicing regulator and the candidate tumor suppressor gene. Together with previous studies, our work suggests that splicing control of cell death genes may be an important aspect in tumorigenesis. Enhancing the expression or activities of splicing regulators that promote the production of proapoptotic splicing isoforms might provide a therapeutic approach to cancer.alternative splicing regulation ͉ cancer ͉ cell death ͉ RNA binding protein

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“…S5B) (27). Interestingly, ZFP106, SF3A2, and ZFR all contain a specific C2H2 ZnF motif (C-X 2 -C-X 12 -H-X 5 -H) that was previously identified to be conserved in Matrin 3 (MATR3) and several other RNA-binding proteins, which differs from the typical DNA-binding C2H2 ZnF motif (Fig.…”

Section: Resultsmentioning

confidence: 88%

Severe muscle wasting and denervation in mice lacking the RNA-binding protein ZFP106

Anderson

Cannavino

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Innervation of skeletal muscle by motor neurons occurs through the neuromuscular junction, a cholinergic synapse essential for normal muscle growth and function. Defects in nerve-muscle signaling cause a variety of neuromuscular disorders with features of ataxia, paralysis, skeletal muscle wasting, and degeneration. Here we show that the nuclear zinc finger protein ZFP106 is highly enriched in skeletal muscle and is required for postnatal maintenance of myofiber innervation by motor neurons. Genetic disruption of Zfp106 in mice results in progressive ataxia and hindlimb paralysis associated with motor neuron degeneration, severe muscle wasting, and premature death by 6 mo of age. We show that ZFP106 is an RNA-binding protein that associates with the core splicing factor RNA binding motif protein 39 (RBM39) and localizes to nuclear speckles adjacent to spliceosomes. Upon inhibition of pre-mRNA synthesis, ZFP106 translocates with other splicing factors to the nucleolus. Muscle and spinal cord of Zfp106 knockout mice displayed a gene expression signature of neuromuscular degeneration. Strikingly, altered splicing of the Nogo (Rtn4) gene locus in skeletal muscle of Zfp106 knockout mice resulted in ectopic expression of NOGO-A, the neurite outgrowth factor that inhibits nerve regeneration and destabilizes neuromuscular junctions. These findings reveal a central role for Zfp106 in the maintenance of nerve-muscle signaling, and highlight the involvement of aberrant RNA processing in neuromuscular disease pathogenesis.ZNF106 | amyotrophic lateral sclerosis (ALS) | neuromuscular junction (NMJ) | motor neuron disease (MND) | pre-mRNA splicing S ignaling between the presynaptic terminus of a motor neuron and the postsynaptic membrane of a skeletal myofiber occurs at the neuromuscular junction (NMJ), the site of communication that allows for neural control of myofiber identity, growth, and contractility (1). Motor neuron damage or perturbation of nervemuscle signaling through disruption of the NMJ causes a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), spinal muscular atrophy (SMA), and hereditary spastic paraplegia (HSP) (2-6). These neurodegenerative diseases are genetically heterogeneous, with mutations in proteins associated with diverse cellular and molecular functions (7)(8)(9)(10). Although the precise disease mechanisms underlying the pathogenesis of these disorders remain unclear, abnormalities in apoptotic signaling, oxidative stress, protein folding, and RNA processing in motor neurons and skeletal muscle have been implicated in these diseases (11)(12)(13)(14)(15).In a bioinformatics screen for nuclear proteins with enriched expression in skeletal muscle, we identified ZFP106, a zinc finger protein first characterized as an immunodominant cytotoxic determinant of the mouse H3 minor histocompatibility complex (16,17). Zinc fingers (ZnFs) mediate DNA, RNA, and protein interactions and are commonly found in regulatory proteins that govern gen...

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Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Cited by 108 publications

References 100 publications

Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5

Severe muscle wasting and denervation in mice lacking the RNA-binding protein ZFP106

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