A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing

Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3′-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module. The composite double-stranded /single-stranded RNA-binding region assembles cooperatively with Snu13 and Prp31 on U4/U6 di-snRNAs and inhibits Brr2-mediated U4/U6 di-snRNA unwinding in vitro. RNP-disrupting mutations in Prp3 lead to U4/U6•U5 tri-snRNP assembly and splicing defects in vivo. Our results reveal how Prp3 acts as an important bridge between U4/U6 and U5 in the tri-snRNP and comparison with a Prp24-U6 snRNA recycling complex suggests how Prp3 may be involved in U4/U6 reassembly after splicing

Untersuchungen der Protein-Protein-Interaktionen innerhalb des humanen spleißosomalen U4/U6.U5 tri-snRNP-Partikels

Daten aus two-hybrid-Versuchen erlaubten es mir, Regionen zu definieren, über die die Proteine 220K, 200K und 116K miteinander interagieren. Diese Regionen beinhalten die N-terminale und C-terminale Region des 220K Proteins, die zweite Helikase-Domäne des 200K und die C-terminale Domäne des 116K Proteins. Interessanterweise wurden die meisten dieser Bindungsdomänen für die Interaktionen zwischen den Hefe-Orthologen Prp8p, Brr2p und Snu114p identifiziert.Das 102K Protein interagiert mit mehreren Partikel-spezifischen Proteinen des U5, wie auch des U4/U6 snRNPs. Es kann daher als potentiell brückenbildendes oder vernetzendes Protein bei der Formation oder der strukturellen Stabilisierung des tri-snRNPs gesehen werden. Die stabile Integration des 102K Proteins in das U5 snRNP wird über Interaktionen mit dem U5-220K, 200K und dem 116K Protein vermittelt. In dieser Arbeit wurde das U5-spezifische 102K Protein als einziges Protein identifiziert, welches mit den U4/U6-spezifischen Proteinen 61K und 90K interagiert. Es konnte gezeigt werde, daß die Interaktion zwischen 102K und 61K wichtig für die Formierung des tri-snRNPs ist. Das Krankheitsbild der Retinitis pigmentosa entsteht durch eine missense-Mutationen (A194E) im 61K Protein. In dieser Studie zeigte das mutierte Protein 61K eine schlechtere Bindung zum 102K. Die möglichen Mechanismen werden diskutiert. Mutationsanalysen zeigten, daß die TPR-Wiederholungen im 102K Protein jeweils spezifischen Interaktionspartnern zugeschrieben werden können. Alle TPR-Wiederholungen partizipieren in der Interaktion mit dem U4/U6-61K, wohingegen nur die ersten neun Wiederholungen mit dem 110K Protein, dem 200-4 Fragment des 200K und dem 220-1 Fragment des 220K Proteins interagieren.Das U4/U6-90K Protein interagiert innerhalb des 20K 60K 90K-Heterotrimers mit dem 60K Protein und kontaktiert die Stamm-II-Region der U6 snRNA im U4/U6-snRNP. In dieser Studie wurde gezeigt, daß das 90K Protein mit dem humanen recycling factor U6-p110 interagiert und daher eine Funktion im Recycling des U4/U6 snRNPs vermuten läßt. Mutationsanalysen haben gezeigt, daß der C-Terminus des 90K Proteins (Aminosäuren 417-683) verantwortlich für die Bindung zum U6-p110 ist. Während der Formierung des tri-snRNPs interagiert das U5-102K Protein ebenfalls mit dem C-Terminus des 90K Proteins, was vermuten läßt, daß das U5-102K Protein eine Rolle bei der Freisetzung des U6-p110 spielt. Weiter interagiert das 90K Protein über die N-terminale Region mit dem U2 snRNP-assoziierten Protein SPF30/SMNrp und könnte daher bei der Rekrutierung des tri-snRNPs in das Präspleißosom wichtig sein.Das tri-snRNP-spezifische Protein 110K interagiert mit dem U4/U6-90K, dem U5-102K und dem U5-200K Protein über seine C-terminale Region (ohne RS-Domäne). Da die Abwesenheit des 110K Proteins keinen Einfluß auf die Stabilität des tri-snRNPs hat, scheint eher die genaue Positionierung des 110K für die Unterstützung der Bindung des tri-snRNPs mit dem Präspleißosom wichtig zu sein.Das U5-52K Protein interagiert mit dem U5-102K und dem 15K Protein, wobei diese Interaktionen vermutlich die Integration des U5-52K in das U5 snRNP unterstützen. Bindungsstudien, die mit Deletionsmutanten des 52K Proteins durchgeführt wurden, zeigten, daß die N-terminalen zwei Drittel des 52K Proteins mit dem 102K interagieren und die C-terminale GYF-Domäne das 15K Protein bindet. Die GYF-Domäne wurde kürzlich als Polyprolin-bindendes Molekül beschrieben. Da das 15K Protein keine Prolin-reiche Sequenz aufweist, zeigen diese Daten zum ersten Mal, daß eine GYF-Domäne auch in einer Polyprolin-unabhängigen Weise an spezifischen Protein-Protein-Interaktionen beteiligt sein kann. Die kristallographischen Studien der GYF-Domäne des 52K Proteins im Komplex mit dem 15K Protein, die in einer kooperativen Arbeit mit dem Labor von Prof. Dr. Ralf Ficner an der Universität Göttingen gemacht wurden, zeigten, daß das 15K Protein Kontakte mit einem anderen Bereich der GYF-Domäne eingeht, als dies bei Prolin-reichen Molekülen der Fall ist. Daten aus dieser Studie und anderen zeigen, daß das 52K Protein als einziges, U5-spezifisches Protein nicht in das tri-snRNP integriert wird.Auf der Basis der Daten dieser Arbeit schlage ich ein Model für die Assemblierung des U4/U6.U5 tri-snRNPs vor

Insertions and Appendices of the Jab1/MPN Domain of Other Eukaryotic Jab1/MPN Domain Proteins are Functionally Similar but Structurally Distinct from Prp8

We have shown here that the Jab1/MPN-like domain embodies an important protein-protein interaction platform in Prp8 orthologs, primarily due to inserted and appended structural elements. Elements outside the Jab1/MPN core apparently also sustain adaptor functions in other family members (Fu et al., 2001). Indeed, these additions to the core may generally serve to incorporate Jab1/MPN proteins into multi-subunit macromolecular machineries. Sequence alignments reveal some aromatic and aliphatic residues in similar positions in the appendices, suggesting that they could adopt similar structures and attach to the Jab1/MPN core in a similar fashion as seen in scPrp8p 2147-2397. On the other hand, certain evolutionary remodeling events, such as conversion of the substrate channel to a permanent binding site for an inserted element, seem unlikely for Jab1/MPN domain proteins that remain enzymatically active. We used established bioinformatics tools to address the question, whether all expanded Jab1/MPN domains structurally conform to a homologous 'macro'-Jab1/MPN domain as represented by scPrp8p 2147-2397. The sequences of selected proteins (scRpn11p, hCsn5, hBRCA1/BRCA2-containing complex subunit 3 [hBRCC3], and subunit 5ε of heIF3) were submitted to the GeneSilico metaserver (Kurowski and Bujnicki, 2003) to predict their secondar

Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6·U5 tri-snRNP formation and pre-mRNA splicing

In each round of nuclear pre-mRNA splicing, the U4/U6·U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6·U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing. Significantly, complementation with recombinant 61K protein restores each of these steps. Protein 61K is operationally defined as U4/U6 snRNP-specific as it remains bound to this particle at salt concentrations where the tri-snRNP dissociates. However, as shown by two-hybrid analysis and biochemical assays, protein 61K also interacts specifically with the U5 snRNP-associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the tri-snRNP. Interestingly, protein 61K is encoded by a gene (PRPF31) that has been shown to be linked to autosomal dominant retinitis pigmentosa. Thus, our studies suggest that disruptions in tri-snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease

Ganxiao Dong: A Hotspot of Cave Biodiversity in Northern Guangxi, China

Located in the core zone of Mulun National Nature Reserve in northern Guangxi, the limestone cave Ganxiao Dong harbours the richest cave fauna currently known in China. In total, 26 species of cave invertebrates have been recognized so far, in spite of limited sampling efforts. Of them, 20 are troglobionts or stygobionts, including one snail, four millipedes, three spiders, one harvestman, three isopods, two springtails, two crickets, one non-glowing sticky worm, and three trechine beetles. Six other species are troglophiles. The most remarkable characteristic of this fauna is its high number of troglomorphic species, especially among millipedes, crickets and beetles

The human U5 snRNP 52K protein (CD2BP2) interacts with U5-102K (hPrp6), a U4/U6.U5 tri-snRNP bridging protein, but dissociates upon tri-snRNP formation

The U5 snRNP plays an essential role in both U2- and U12-dependent splicing. Here, we have characterized a 52-kDa protein associated with the human U5 snRNP, designated U5-52K. Protein sequencing revealed that U5-52K is identical to the CD2BP2, which interacts with the cytoplasmic portion of the human T-cell surface protein CD2. Consistent with it associating with an snRNP, immunofluorescence studies demonstrated that the 52K protein is predominantly located in the nucleoplasm of HeLa cells, where it overlaps, at least in part, with splicing-factor compartments (or “speckles”). We further demonstrate that the 52K protein is a constituent of the 20S U5 snRNP, but is not found in U4/U6.U5 tri-snRNPs. Thus, it is the only 20S U5-specific protein that is not integrated into the tri-snRNP and resembles, in this respect, the U4/U6 di-snRNP assembly factor Prp24p/p110. Yeast two-hybrid screening and pulldown assays revealed that the 52K protein interacts with the U5-specific 102K and 15K proteins, suggesting that these interactions are responsible for its integration into the U5 particle. The N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. As the latter lacks a proline-rich tract, our data indicate that a GYF-domain can also engage in specific protein–protein interactions in a polyproline-independent manner. Interestingly, the U5-102K protein has been shown previously to play an essential role in tri-snRNP formation, binding the U4/U6-61K protein. The interaction of 52K with a tri-snRNP bridging protein, coupled with its absence from the tri-snRNP, suggests it might function in tri-snRNP assembly

Feihu Dong, a New Hotspot Cave of Subterranean Biodiversity from China

China is a country with abundant karst landscapes, but research on cave biodiversity is still limited. Currently, only Ganxiao Dong, located in Huanjiang, Guangxi, has been reported as a hotspot for cave biodiversity. Many of the world’s most troglomorphic species in the major groups of cave animals have been recently discovered in China, making the existence of many more hotspots in the country likely. Feihu Dong, one of these potential hotspot caves, has been systematically investigated to complement a preliminary species list of 1995, leading to the discovery of 62 species of animals from the cave. Among them, 27 are considered troglobionts or stygobionts, 26 are considered troglophiles or stygophiles, and nine are classified as trogloxenes or stygoxenes. Research on the cave biodiversity of Feihu Dong has demonstrated that it currently holds the highest number of known cave animal species in China. Among the most remarkable features of this fauna is the co-occurrence of five species of cave-obligate beetles, all modified for cave life. The biological survey was limited to a small part of the cave. Several habitats (like guano) have not been investigated so far, and several important cave groups have been insufficiently or not sampled (like Ostracoda). Meanwhile, the system increases in length with each new caving expedition. Further discoveries of cave organisms in Feihu Dong are therefore expected. As Feihu Dong and Ganxiao Dong are the only caves in China that have been extensively studied for a large range of organisms, and as they are located in karstic areas that are similar in richness to other regions of southern China, it can be confidently assumed that several other caves of high biodiversity will be discovered in the coming years