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...

Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 Å, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5′-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.Fluorescence resonance energy transfer (FRET) has been applied to the analysis of global conformation and folding transitions in nucleic acids with increasing frequency in the past decade. Potentially, the method provides distance information between known points that is typically in the range of 10-80 Å. This is a range that is inaccessible to other solution methods, and thus, the approach is complementary in principle to NMR in particular. FRET has been applied to the analysis of DNA structures (1-10) and more recently to the folding of a number of interesting RNA structures (11-18).Determination of either relative or absolute distance information using FRET depends on the distance-dependent transfer of excitation energy from a donor to an acceptor fluorophore, resulting from the coupling between their transition dipoles. The efficiency of the transfer (E FRET ) depends inversely on the sixth power of the distance separating the fluorophores (19), and is given by where R is the scalar distance between the fluorophores and R 0 is the characteristic Förster distance for the donoracceptor combination at which E FRET equals 0.5. In general in our studies, we attach the fluorophores to the 5′-termini of known helical arms with a constant terminal sequence, and in this way, we can compare different end-to-end distances and study how these change upon the induction of conformational change.Most FRET studies of nucleic acids have used fluorescein as the donor, and many have used tetramethylrhodamine as the acceptor. Fluorescein is generally very mobile when attached to DNA or RNA, and thus, orientation is not a problem in the interpretation of the measured FRET efficiencies. However, tetramethylrhodamine has some disadvantages, including the difficulty of coupling the fluorophore to the nucleic acid as a phosphoramidite. For this reason, we have largely changed to the use of indocarbocyanine-3 (Cy3) as the acceptor in our recent studies (F...

Prp8 protein is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 snRNPs. Intriguingly, although it is ubiquitously expressed, mutations in the C-terminus of human Prp8p cause the retina-specific disease Retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks a mature U5 snRNP component, Brr2p, and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes, which is disrupted by mutations that cause human RP.Nuclear pre-mRNA splicing is an essential housekeeping process in all eukaryotic cells. It is catalyzed by a large ribonucleoprotein (RNP) complex called the spliceosome, which contains the small nuclear RNPs (snRNPs) U1, U2, U4, U5 and U6, as well as many nonsnRNP proteins1, 2. Each snRNP consists of an snRNA, a set of specific proteins, and seven common Sm proteins or, in the case of U6 snRNP, seven Lsm proteins.Unexpectedly, mutations in four human snRNP-associated proteins, PRPF83, PRPF314, PRPF35 and PAP-1/RP96, 7 were found in patients with a dominantly inherited form of retinal degeneration, Retinitis pigmentosa (RP). Here, we investigate the role of Prp8p (the yeast ortholog of PRPF8) in U5 snRNP biogenesis in Saccharomyces cerevisiae, and the effect of RP mutations on this process.Biogenesis of the U snRNPs has been studied extensively in metazoans1, 8. The U1, U2, U4 and U5 snRNAs are produced as precursors in the nucleus by RNA polymerase II then exported to the cytoplasm, facilitated by nuclear cap-binding proteins and the export factors, CRM1 and PHAX8. In the cytoplasm seven Sm proteins bind to the snRNAs, facilitated by the SMN complex9, 10, and the m 7 G cap is hypermethylated to form a 2,2,7-