Biomedical subjects

Reinhard Lührmann

Publications and source records attributed to Reinhard Lührmann.

At least 37 records · Page 2Linked to original sources

PubMed2005

The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11-59K protein.

U11 and U12 interact cooperatively with the 5' splice site and branch site of pre-mRNA as a stable preformed di-snRNP complex, thereby bridging the 5' and 3' ends of the intron within the U12-dependent prespliceosome. To identify proteins contributing to di-snRNP formation and intron bridging, we investigated protein-protein and protein-RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12-65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C-terminal RRM and the U11-associated 59K protein via its N-terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3' half of U12 forms an extended stem-loop with a highly conserved seven-nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12-dependent, but not U2-dependent, pre-mRNA splicing. Taken together, these data suggest that U11/U12-65K and U11-59K contribute to di-snRNP formation and intron bridging in the minor prespliceosome.

Amino Acid Motifs↗

PubMed2005

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1.

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.

Active Transport, Cell Nucleus↗

PubMed2005

Major conformational change in the complex SF3b upon integration into the spliceosomal U11/U12 di-snRNP as revealed by electron cryomicroscopy.

In some eukaryotes, a minor class of introns is removed by the U12-dependent spliceosome, which contains the small nuclear ribonucleoprotein (snRNP) heterodimer U11/U12. The U11/U12 di-snRNP forms a molecular bridge that functionally pairs the intron ends of the pre-mRNA. We have determined the three-dimensional (3D) structure of the human U11/U12 di-snRNP by single particle electron cryomicroscopy using angular reconstitution and random conical tilt. SF3b, a heteromeric protein complex functionally important for branch site recognition, was located in the U11/U12 di-snRNP by antibody labeling and by identification of structural domains of SF3b155, SF3b49, and p14. The conformation of SF3b bound to the U11/U12 di-snRNP differs from that of isolated SF3b: upon integration into the di-snRNP, SF3b rearranges into a more open form. The manner in which SF3b is integrated in the U11/U12 di-snRNP has important implications for branch site recognition. Furthermore, a putative model of the pre-mRNA binding to the U11/U12 di-snRNP is proposed.

Cryoelectron Microscopy↗

PubMed2005

Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.

Protein-RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto non-characterized protein-RNA contacts in ribonucleoprotein particles, as, for example, within spliceosomes. Here, we describe an improved methodology for the sequence analysis of purified peptide-RNA oligonucleotide cross-links that is based solely on MALDI-ToF mass spectrometry. The utility of this methodology is demonstrated on cross-links isolated from UV-irradiated spliceosomal particles; these were (1) [15.5K-61 K-U4 atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro, and (2) U1 snRNP particles purified from HeLa cells. We show that the use of 2',4',6'-trihydroxyacetophenone (THAP) as MALDI matrix allows analysis of cross-linked peptide-RNA oligonucleotides in the reflectron mode at high resolution, enabling sufficient accuracy to assign unambiguously cross-linked RNA sequences. Most important, post-source decay (PSD) analysis under these conditions was successfully applied to obtain sequence information about the cross-linked peptide and RNA moieties within a single spectrum, including the identification of the actual cross-linking site. Thus, in U4 atac snRNA we identified His 270 in the spliceosomal U4/U6 snRNP-specific protein 61 K (hPrp31p) cross-linked to U 44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U 30 of U1 snRNA. This type of analysis is applicable to any type of RNP complex and may be expected to pave the way for the further analysis of protein-RNA complexes in much lower abundance and/or of cross-links that are obtained in low yield.

Amino Acid Sequence↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies.

mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly understood. We identified protein factors potentially involved in this transition, which are present in mammalian P bodies, cytoplasmic foci enriched in 5' --> 3' mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs for 5' --> 3' degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed that mRNA is also required for accumulation of mRNA degradation factors in P bodies. In contrast, knockdown of the decapping enzyme Dcp2, which initiates the actual 5' --> 3' mRNA degradation did not abolish P body formation, indicating it first functions after mRNPs have been targeted to these cytoplasmic foci. These data support a model in which mRNPs undergo several successive steps of remodeling and/or 3' trimming until their composition or structural organization promotes their accumulation in P bodies.

Biological Transport↗

PubMed2005

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements.

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.

Computer Simulation↗

PubMed2005

Splicing of a rare class of introns by the U12-dependent spliceosome.

Pre-mRNA splicing is catalyzed by two unique spliceosomes, designated U2- or U12-dependent. In contrast to the well-characterized U2-dependent spliceosome, much remains to be learned about the less abundant U12-type spliceosome. This review focuses on recent advances in elucidating the structure and function of the minor U12-dependent spliceosome. Interesting similarities and differences between the U12- and U2-dependent spliceosomes are also highlighted.

Amino Acid Sequence↗

PubMed2004

Assembly and maturation of the U3 snoRNP in the nucleoplasm in a large dynamic multiprotein complex.

The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.

Base Sequence↗

PubMed2004

Protein stoichiometry of a multiprotein complex, the human spliceosomal U1 small nuclear ribonucleoprotein: absolute quantification using isotope-coded tags and mass spectrometry.

The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

Chromatography, Liquid↗

PubMed2004

Modified nucleotides at the 5' end of human U2 snRNA are required for spliceosomal E-complex formation.

U2 snRNA, a key player in nuclear pre-mRNA splicing, contains a 5'-terminal m3G cap and many internal modifications. The latter were shown in vertebrates to be generally required for U2 function in splicing, but precisely which residues are essential and their role in snRNP and/or spliceosome assembly is presently not clear. Here, we investigated the roles of individual modified nucleotides of HeLa U2 snRNA in pre-mRNA splicing, using a two-step in vitro reconstitution/complementation assay. We show that the three pseudouridines and five 2'O-methyl groups within the first 20 nucleotides of U2 snRNA, but not the m3G cap, are required for efficient pre-mRNA splicing. Individual pseudouridines were not essential, but had cumulative effects on U2 function. In contrast, four of five 2'O-methylations (at positions 1, 2, 12, and 19) were individually required for splicing. The in vitro assembly of 17S U2 snRNPs was not dependent on the presence of modified U2 residues. However, individual internal modifications were required for the formation of the ATP-independent early spliceosomal E complex. Our data strongly suggest that modifications within the first 20 nucleotides of U2 play an important role in facilitating the interaction of U2 with U1 snRNP and/or other factors within the E complex.

Base Sequence↗

PubMed2004

Coupled in vitro import of U snRNPs and SMN, the spinal muscular atrophy protein.

Cytoplasmic assembly of Sm-class small nuclear ribonucleoproteins (snRNPs) is a central process in eukaryotic gene expression. A large macromolecular complex containing the survival of motor neurons (SMN) protein is required for proper snRNP assembly in vivo. Defects in SMN function lead to a human neuromuscular disorder, spinal muscular atrophy (SMA). SMN protein localizes to both nuclear and cytoplasmic compartments, and a reduction in nuclear levels of SMN is correlated with the disease. The mechanism of SMN nuclear import, however, is unknown. Using digitonin-permeabilized cells, we show that SMN import depends on the presence of Sm snRNPs. Conversely, import of labeled U1 snRNPs was SMN complex dependent. Thus, import of SMN and U snRNPs are coupled in vitro. Furthermore, we identify nuclear import defects in SMA patient-derived SMN mutants, uncovering a potential mechanism for SMN dysfunction.

Carrier Proteins↗

PubMed2004

Purification, crystallization and preliminary crystallographic data of the m3G cap-binding domain of human snRNP import factor snurportin 1.

The nuclear import of spliceosomal UsnRNPs is mediated by the transport adaptor snurportin 1 (SPN1), which specifically recognizes the 2,2,7-trimethylguanosine (m(3)G) cap at the 5' end of UsnRNAs. Human SPN1 was overexpressed as a GST-fusion protein in Escherichia coli and purified to homogeneity. Since full-length SPN1 did not crystallize, limited proteolysis experiments were performed and stable digestion products were analyzed for functionality with respect to m(3)G cap-binding activity and subsequently used for crystallization trials. Well diffracting single crystals of a truncated SPN1 m(3)G cap-binding domain (residues 79-300) were obtained after two rounds of seeding. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 57.47, c = 130.09 A, alpha = beta = gamma = 90 degrees. Crystals contain one molecule in the asymmetric unit and diffract to a resolution limit of 2.9 A.

Cross-Linking Reagents↗

PubMed2004

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies.

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.

Carrier Proteins↗

PubMed2004

Identification of an evolutionarily divergent U11 small nuclear ribonucleoprotein particle in Drosophila.

Previous reports suggested that U11, in contrast to U12 or other small nuclear (sn)RNAs of the U12-type spliceosome, might be either highly divergent or absent in Drosophila melanogaster. Affinity purification of Drosophila U12-containing complexes has led to the identification of the fly U11 snRNA, which contains a potential U12-type 5' splice-site-interacting sequence, but whose sequence and length differs significantly from vertebrate and plant U11. Analysis of U12-type introns revealed an A-rich region directly downstream of Drosophila, but not human, U12-type 5' splice sites. This finding, coupled with the presence of a highly divergent U11 snRNA, and the apparent absence of Drosophila homologs of human U11 proteins, suggest that U12-type 5' splice site recognition might be different in flies. A comparison of U11 snRNAs that we have identified from vertebrates, plants, and insects, suggests that an evolutionarily divergent U11 snRNA may be unique to Drosophila and not characteristic of insects in general.

Animals↗

PubMed2004

A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing.

During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human spliceosomes that had not yet undergone catalytic activation (designated BDeltaU1), which contained U2, U4, U5, and U6, but lacked U1 snRNA. Comparison of the protein compositions of BDeltaU1 and activated B* spliceosomes revealed that, whereas U4/U6 snRNP proteins are stably associated with BDeltaU1 spliceosomes, 35S U5-associated proteins (which are present in B*) are largely absent, suggesting that they are dispensable for complex B formation. Indeed, immunodepletion/complementation experiments demonstrated that a subset of 35S U5 proteins including Prp19, which form a stable heteromeric complex, are required prior to catalytic step 1 of splicing, but not for stable integration of U4/U6.U5 tri-snRNPs. Thus, comparison of the proteomes of spliceosomal complexes at defined stages can provide information as to which proteins function as a group at a particular step of splicing.

Carrier Proteins↗

PubMed2004

Three-dimensional structure of a pre-catalytic human spliceosomal complex B.

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated B Delta U1) at a stage after U4/U6.U5 tri-snRNP integration but before activation, and have determined the three-dimensional structure of B Delta U1 by single-particle electron cryomicroscopy at a resolution of approximately 40 A. The overall size of the complex is about 370 x 270 x 170 A. The three-dimensional structure features a roughly triangular body linked to a head domain in variable orientations. The body is very similar in size and shape to the isolated U4/U6.U5 tri-snRNP. This provides initial insight into the structural organization of complex B.

Catalysis↗

PubMed2004

Human U4/U6 snRNP recycling factor p110: mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling.

After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

Animals↗