Crystal structure of the C‐terminal domain of splicing factor Prp8 carrying retinitis pigmentosa mutants

Abstract

Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an a/b structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn 2+ coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn 2+ -dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.Keywords: Prp8; retinitis pigmentosa; MPN domain; crystal structure Supplemental material: see www.proteinscience.org Pre-mRNA splicing is carried out by the spliceosome, a huge RNA/protein complex made of five snRNAs and over 100 protein factors. Prp8 is a key factor for premRNA splicing. Prp8 was proposed to help form/stabilize the catalytic core and to be an important regulator in spliceosome activation (Collins and Guthrie 2000; Grainger and Beggs 2005). Prp8 is the only spliceosome protein that extensively cross-links with the 59 splice site, the 39 splice site, the branch point sequence, and the U5 and U6 snRNAs, all of which are considered components of the catalytic core (Grainger and Beggs 2005 and references therein). In addition, Prp8 interacts with a number of protein partners required for spliceosome activation (Grainger and Beggs 2005 and references therein; Brenner and Guthrie 2006;Liu et al. 2006;Small et al. 2006). Many Prp8 mutations suppress or enhance splicing defects caused by splice-site mutations and other mutations affecting spliceosome activation or the second step of splicing (Grainger and Beggs 2005 and references therein). Several mutations in the C terminus of Prp8 are associated with RP13, a severe form of human genetic disorder retinitis pigmentosa, which leads to progressive photoreceptor degeneration in the retina and eventual blindness (McKie et al. 2001;van Lith-Verhoeven et al. 2002;Kondo et al. 2003;Martinez-Gimeno et al. 2003).Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been difficult due to its large size and low-sequence similarity 3 These authors contributed equally to this work. Reprint requests to: Rui Zhao, M.S. 8101, P.O. Box 6511, Aurora, CO 80045, USA; e-mail: rui.zhao@uchsc.edu; fax: (303) 724-3215.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/...

Crystal structure of the β-finger domain of Prp8 reveals analogy to ribosomal proteins

“…We determined the crystal structure of the ␤-finger domain (residues 1,822-2,095) of yPrp8 to 2.05-Å resolution. The ␤-finger domain is immediately upstream of the C-terminal domain (CTD, residues 2,143-2,413) structure we determined earlier (13,14) (Fig. 1A).…”

“…The C-terminal domain structure we determined earlier (13,14) has the MPN fold present in several Zn 2ϩ -dependent isopep- , and Caenorhabditis elegans Prp8 (cPrp8) in the ␤-finger domain region. Alignment was performed by using MultAlin (40).…”

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA

“…Ubiquitination of Prp8 has been proposed to play a role in regulating the activity of Brr2, but the mechanism whereby this is achieved is currently unknown (Bellare et al 2008). A potential target is the C terminus of Prp8, which contains a Jab1/MPN-like domain (Pena et al 2007;Zhang et al 2007) that has lost its deubiquitination activity but retained its ability to bind ubiquitin (Bellare et al 2006). The Prp8 CTF also contains a domain with a threedimensional (3D) fold resembling RNase H (RH), flanked on one side by a b-hairpin loop and on the other by an a-helical domain (Pena et al 2008;Ritchie et al 2008;Yang et al 2008).…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…Interestingly, the Prp8 Jab1 domain resembles corresponding domains in deubiquitinating enzymes. 85,86 Although it is catalytically inactive as a deubiquitinase, it can still bind ubiquitin. 120 Thus, it is conceivable that ubiquitinated spliceosomal proteins contact the Jab1 domain via their ubiquitin units and influence its ability to inhibit or activate Brr2.…”