Cwc23, an Essential J Protein Critical for Pre-mRNA Splicing with a Dispensable J Domain

Abstract

J proteins are structurally diverse, obligatory cochaperones of Hsp70s, each with a highly conserved J domain that plays a critical role in the stimulation of Hsp70's ATPase activity. The essential protein, Cwc23, is one of 13 J proteins found in the cytosol and/or nucleus of Saccharomyces cerevisiae. We report that a partial loss-of-function CWC23 mutant has severe, global defects in pre-mRNA splicing. This mutation leads to accumulation of the excised, lariat form of the intron, as well as unspliced pre-mRNA, suggesting a role for Cwc23 in spliceosome disassembly. Such a role is further supported by the observation that this mutation results in reduced interaction between Cwc23 and Ntr1 (SPP382), a known component of the disassembly pathway. However, Cwc23 is a very atypical J protein. Its J domain, although functional, is dispensable for both cell viability and pre-mRNA splicing. Nevertheless, strong genetic interactions were uncovered between point mutations encoding alterations in Cwc23's J domain and either Ntr1 or Prp43, a DExD/H-box helicase essential for spliceosome disassembly. These genetic interactions suggest that Hsp70-based chaperone machinery does play a role in the disassembly process. Cwc23 provides a unique example of a J protein; its partnership with Hsp70 plays an auxiliary, rather than a central, role in its essential cellular function.Hsp70-based machineries constitute a key component of the cell's chaperone network, playing a central role in many processes, including de novo protein folding, protein translocation across membranes, and remodeling of protein complexes (6, 13). By their ability to bind to short, exposed, hydrophobic stretches of polypeptide, Hsp70s serve as the core of this protein folding machinery. However, Hsp70s cannot function alone. J proteins (often referred to as Hsp40s) are their obligatory partners, serving to stimulate Hsp70's ATPase activity and thereby stabilizing interaction with client proteins (19). J proteins are very diverse but, by definition, contain an ϳ70-amino-acid "J domain" that interacts directly with the Hsp70 ATPase domain (12,17). It is well established that the J domain is critical for function of the Hsp70-based chaperone machinery, since single amino acid alterations in its highly conserved HPD motif disrupt function, both in vitro and in vivo, without affecting domain structure (9, 14). The Saccharomyces cerevisiae genome encodes 22 J proteins. Of these, 13 are found in the cytosol and/or nucleus. One, Cwc23, is the focus of this report. Deletion of CWC23 causes inviability in some strain backgrounds, although adaptation or suppression allows slow growth in others (31, 34). The growth defect of cwc23⌬ cells cannot be rescued by overexpression of any of the other 12 J proteins (31), indicating that Cwc23 carries out a specialized cellular function. Cwc23 has been linked to premRNA splicing via both genome-wide and spliceosomal component-directed physical interaction studies (7,25).Pre-mRNA splicing is a highly precise and stepwise process wher...

The function of the NineTeen Complex (NTC) in regulating spliceosome conformations and fidelity during pre-mRNA splicing

“…Cwc23 is one of 13 J proteins found in S. cerevisiae. A partial loss-of-function Cwc23 mutant displays an increase in unspliced pre-mRNA and an increase in the lariat intron, a phenotype common in mutants which affect spliceosome disassembly [30,31]. Furthermore, deletion of the Cwc23 J domain is synthetically lethal with mutations that compromise the interaction between Ntr1 and Prp43 [30].…”

“…The non-essential NTC-associated protein Cwc23 has also been implicated in spliceosome disassembly through its interaction with Ntr1 [29,30]. Cwc23 is one of 13 J proteins found in S. cerevisiae.…”

“…A partial loss-of-function Cwc23 mutant displays an increase in unspliced pre-mRNA and an increase in the lariat intron, a phenotype common in mutants which affect spliceosome disassembly [30,31]. Furthermore, deletion of the Cwc23 J domain is synthetically lethal with mutations that compromise the interaction between Ntr1 and Prp43 [30]. These results imply that the J domain is important for the function of the NTR complex; however, as the J domain is itself dispensable, its function may be important under different environmental conditions [30].…”

Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1

“…i Western analysis of total protein lysate prepared from djp1Δ cells harboring either CEN-ADHDjp1 or CEN-ADH-Djp1 (ΔC50) plasmid was done as mentioned above of Djp1, the J domain of Caj1 becomes important for GFP-PTS1 localization in S. cerevisiae. Cwc23 is the only other cytosolic J protein that has a dispensable J domain; however, in this case, a compensatory effect of other J proteins was not found (Sahi et al 2010). Functional overlap between cytosolic J proteins in budding yeast is not new.…”

“…Different cytosolic J protein deletion strains, djp1Δ, caj1Δ, xdj1Δ, ydj1Δ, apj1Δ, hlj1Δ, jjj1Δ, jjj2Δ, jjj3Δ, and swa2Δ, along with the wild-type strain with BY4743 genetic background were obtained from Thermo Yeast Knockout Collection (Open Biosystem). cwc23Δ cells harboring a Cwc23ΔJ plasmid (Sahi et al 2010) and sis1Δ strain (Lopez et al 2003) with Sis1 expressed from a tetracycline repressible promoter (Tet R-SIS1) were utilized to study the function of essential J proteins Cwc23 and Sis1. djp1Δcaj1Δ double deletion strain was generated to study the genetic interaction between Djp1 and Caj1 by mating djp1Δ::KanMX strain with caj1 Δ::LEU2 strain.…”