Tandem affinity purification protocol for isolation of protein complexes from Schizosaccharomyces pombe

Čipák, Luboš; Selicky, Tomas; Jurcik, Jan; Cipakova, Ingrid; Osadska, Michaela; Lukáčová, Veronika; Baráth, Péter; Gregáň, Juraj

doi:10.1016/j.xpro.2022.101137

Cited by 1 publication

(1 citation statement)

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“…Yeast cell powders (40 g) were prepared from frozen cell pellets using SPEX SamplePrep 6770 Freezer/Mill (SPEX SamplePrep, Metuchen, NJ, USA) cooled by liquid nitrogen. Proteins were extracted using IPP150 buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM PMSF and complete protease and phosphatase inhibitors) in a ratio of 1 g of yeast cell powder to 3 mL of IPP150 buffer and affinity purified as described previously [ 43 , 64 ]. Briefly, 500 µL of IgG Sepharose™ 6 Fast Flow beads (GE Healthcare, Uppsala, Sweden) was equilibrated with IPP150 buffer, mixed with protein extract and incubated on rotatory wheel for 2 h at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe

Selicky

Jurcik²,

Mikolaskova³

et al. 2022

IJMS

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Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.

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Section: Methodsmentioning

confidence: 99%