Solution Structure of Human Peptidyl Prolyl Isomerase-like Protein 1 and Insights into Its Interaction with SKIP

Abstract

The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 ؋ 10 ؊7 M for the N-terminal peptide of SKIP-(59 -129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed.Pre-mRNA splicing, the removal of introns from mRNA precursors, is indispensable to the expression of most eukaryotic genes. The splicing of mRNA is catalyzed by spliceosome, a large machine formed by an ordered interaction of several small nuclear ribonucleoproteins (snRNPs), 3 U1, U2, U5, U4/U6, and numerous other less stably associated non-snRNP splicing factors (1, 2). The formation of spliceosome goes through many intermediate stages. The stable intermediate complexes are the A, B, and C complexes. During the spliceosome maturation process, the most decisive step is the conversion from non-active complex B to the catalytically active spliceosome B*. The activated complex B* undergoes the first catalytic step of splicing and then forms complex C. Prior to the activation of the spliceosome and during the splicing process, a number of conformational rearrangements take place. Recently, human 45 S activated spliceosome (complex B*) and 35 S U5 snRNP have been isolated by immunoaffinity purification and characterized by mass spectrometry (3). Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition. More than 100 proteins were identified in the 45 S activated spliceosome, 80 of which are known splicing factors. The rest are non-snRNP proteins, including protein SKIP and one peptidyl prolyl isomerase-like protein 1 (PPIL1) (2-4).PPIL1 is a component in 45 S U5 snRNP in activated spliceosome (complex B*) and 35 S snRNP. It was believed to participate in the activation of spliceosome (5). The cDNA of PPIL1 was first cloned from human fetal brain, which encodes 166 amino acid residues. PPIL1 has 41.6% identity to human cyclophilin A (6). It belongs to a novel subfamily of cyclophilins, to...

A Large Intrinsically Disordered Region in SKIP and Its Disorder-Order Transition Induced by PPIL1 Binding Revealed by NMR

“…The N-proximal 180 amino acids of SnwA (ortholog of human SKIP) interact with CypE (ortholog of human PPIL1) in Dictyostelium discoideum (18). Xu et al (17) confirmed that the N-terminal of SKIP (residues 59 -129, SKIPN) is sufficient to associate with PPIL1. Nevertheless, how they bind and the structural basis of their binding are still unknown.…”

“…PPIL1 (166 residues) belongs to a novel subfamily of cyclophilin, which contains a single domain (PPIase domain) and has a SKIP-binding region and a cyclosporin A (inhibitor of peptidylprolyl isomerase)-binding region (17). SKIP recruits PPIL1 into the spliceosome.…”

SKIP counteracts p53-mediated apoptosis via selective regulation of p21Cip1 mRNA splicing

“…At the HIV-1 promoter, SKIP recruits c-Myc and also interacts with the MLL1:Menin histone methyltransferase to promote H3K4 methylation (Brè s et al 2009). Previous studies found that SKIP also binds U2AF35 , the PPIL1 peptidyl-prolyl isomerase Xu et al 2006), and the DExH RNA helicase Prp22 (Gahura et al 2009), which helps release mRNA from the spliceosome (Schwer 2008). SKIP is required for cell survival and stress resistance in plants (Hou et al 2009), and depletion of human SKIP or hPrp22 results in mitotic spindle defects and accumulation in prometaphase (Kittler et al 2004(Kittler et al , 2005, indicating an important role in cell cycle progression.…”

Transmembrane Helix 1 Contributes to Substrate Translocation and Protein Stability of Bile Acid Transporter SLC10A2

“…Cis-trans isomerization of proline-linked peptide bonds is a rate-limiting step in protein folding, catalyzed by peptidylprolyl isomerases (PPIs) including cyclophilin A (CyPA) and FK506-binding protein (FKBP) (30,31). PPI inhibition will likely affect protein levels and potentially reveal the mechanism involved in protein (mis)folding of PPI substrates.…”