Prp8 protein: At the heart of the spliceosome

Abstract

Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalyzed by a large RNA-protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa.Prp8 is unique, having no obvious homology to other proteins; however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8's central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes. NOTE ON NOMENCLATUREIn this review, Prp8 and Prp8p represent the protein product of the wild-type PRP8 gene of Saccharomyces cerevisiae, while prp8-1 is an example of a mutant allele of the PRP8 gene. Human Prp8 protein is designated hPrp8, also known in the literature as PRPF8, PRPC8, p220, and 220K. In some places, to avoid confusion, yPrp8 is used to distinguish the yeast (S. cerevisiae) protein from the human form. sPrp8 SPP42 (Schmidt et al. 1999) or sPrp8 cwf6 (McDonald et al. 1999;Ohi et al. 2002) defines the ortholog in Schizosaccharomyces pombe. Confusingly, the cdc28 gene in S. pombe is also referred to as prp8 as a consequence of its role in both pre-mRNA splicing and cell cycle progression, and there is also a temperature-sensitive allele called prp8-1 that causes accumulation of pre-mRNA upon a shift to nonpermissive temperatures (Lundgren et al. 1996;Imamura et al. 1998). Cdc28 prp8 encodes a 112-kDa DEAH-box protein. This protein is not the ortholog of the U5 snRNP protein of S. cerevisiae that is discussed here.In discussions of pre-mRNA-protein cross-links or mutations that affect intron-exon junctions, 5ЈSS+2 refers to the second intronic base from the 5Ј end of the intron, 5ЈSS-2 is the penultimate base of the 5Ј exon, and 3ЈSS-2 is the penultimate base of the intron. PRE-mRNA SPLICINGPre-mRNA splicing involves two trans-esterification reactions within the highly dynamic spliceosome complex. A vast amount of mainly biochemical data led to a consensus view of an ordered pathway of spliceosome assembly that will be described in outline here (for further details, see Kramer 1996;Burge et al. 1999;Brow 2002). The small nuclear RNA-protein (snRNP) complexes, known as U1, U2, U4, U5, and U6 snRNPs, play key roles. U1 is the first snRNP to associate with pre-mRNA, interacting with the 5Ј splice site (5Ј...

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

“…It is the only spliceosomal protein that extensively cross-links with the 5Ј ss, BPS, 3Ј ss, U5, and U6, all key components of the splicing reaction (6). A large number of mutations in PRP8 were identified that suppress the U4-cs1 cold-sensitive mutant, illustrating the role of Prp8 in the spliceosome activation mediated by U4/U6 unwinding (7)(8)(9).…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…Prp8 is the largest and most highly conserved spliceosomal protein 80 and forms a scaffold, on which the catalytic core of the spliceosome is assembled. 39,81 At its C-terminus, Prp8 comprises an RNase H-like (RH) domain [82][83][84] followed by a Jab1 domain, 85,86 both of which can regulate Brr2 function.…”

Role of the snRNAs in spliceosomal active site

“…Crosslinking experiments have indicated the proximity of Prp8 to both the 5' and 3' splice sites and the branch site, in addition to U6 and U5 snRNAs. 76,77 Thus, it is clear that Prp8 is closely associated with reacting groups in assembled spliceosomes. Further, mutations in Prp8 are known to affect the efficiency of either the first or second step of splicing, especially for suboptimal substrates.…”

“…Further, mutations in Prp8 are known to affect the efficiency of either the first or second step of splicing, especially for suboptimal substrates. 57,76,[78][79][80] This observation is best explained by a hypothesis stating that Prp8 is involved in stabilization of alternative, mutually-exclusive active site conformations that are either poised for catalysis of the branching reaction observed in the spliceosome, the cleavage of the 5' splice site occurs through hydrolysis in this minimal, RNA-only system. This could be due to the absence of a branch site-like substrate, which precludes a branching reaction.…”