Biomedical subjects

Reinhard Lührmann

Publications and source records attributed to Reinhard Lührmann.

At least 55 records · Page 3Linked to original sources

PubMed2004

Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP.

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.

Cell Nucleolus↗

PubMed2004

The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome.

U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5' splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein-protein and protein-RNA interactions contributing to 5' splice site recognition and/or intron bridging appear to differ significantly in the minor versus major prespliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type spliceosome provides insight into potential evolutionary relationships between the major and minor spliceosome.

Amino Acid Sequence↗

PubMed2004

Tobramycin affinity tag purification of spliceosomes.

The ability to isolate native ribonucleoprotein (RNP) particles is of fundamental importance in the study of processes such as pre-messenger RNA (mRNA) processing and translation. We have developed an RNA affinity tag that allows the large-scale preparation of native spliceosomes in a solid-phase assembly scheme. A tobramycin-binding aptamer cotranscriptionally added to the 3' end of the pre-mRNA is used to bind the pre-mRNA to tobramycin immobilized on a matrix. Incubation of the pre-mRNA thus immobilized allows the assembly of spliceosomes, which can be released from the matrix under native conditions by competition with tobramycin. Further density-gradient centrifugation affords highly purified spliceosomes suitable for the characterization of associated proteins by mass spectrometry as well as for studies using biochemical and biophysical methods. Although the method was developed for the preparation of spliceosomes, it is likewise applicable to the preparation of other RNP particles.

Binding Sites↗

PubMed2003

Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing.

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

Amino Acid Sequence↗

PubMed2003

Two protein-protein interaction sites on the spliceosome-associated human cyclophilin CypH.

Cyclophilins are a family of proteins that share a common, highly conserved sequence motif. Cyclophilins bind transiently to other proteins and facilitate their folding. One member of the family, hCypH, is part of the human spliceosomal [U4/U6.U5] tri-snRNP complex; it associates specifically and stably with the U4/U6-specific protein 60K. Here, we demonstrate that recombinant hCypH exhibits peptidyl-prolyl isomerase (PPIase) activity, and describe mutagenesis studies demonstrating that it shares the catalytic pocket with other members of the cyclophilin family. However, neither the PPIase activity nor the catalytic pocket is required for binding of protein 60K. Rather, hCypH contains a small insertion in a loop of the otherwise conserved cyclophilin backbone, and this minor change creates a highly specific binding site that is responsible for the association of this cyclophilin, but not others, with protein 60K. hCypH is thus the first small cyclophilin shown to have a second protein-protein interaction site and the ability to bind stably to another protein. Since the catalytic pocket and the second binding site are located on opposite sides of the cyclophilin structure, this opens up the interesting possibility that hCypH may serve as a bridge mediating interactions between protein 60K of the U4/U6 snRNP and other as yet unknown factors.

Binding Sites↗

PubMed2003

Crystal structure of a complex between human spliceosomal cyclophilin H and a U4/U6 snRNP-60K peptide.

The spliceosomal cyclophilin H is a specific component of the human U4/U6 small nuclear ribonucleoprotein particle, interacting with homologous sequences in the proteins U4/U6-60K and hPrp18 during pre-mRNA splicing. We determined the crystal structure of the complex comprising cyclophilin H and the cognate domain of U4/U6-60K. The 31 amino acid fragment of U4/U6-60K is bound to a region remote from the cyclophilin active site. Residues Ile118-Phe121 of U4/U6-60K expand the central beta-sheet of cyclophilin H and the side-chain of Phe121 inserts into a hydrophobic cavity. Concomitantly, in the crystal the cyclophilin H active site is occupied by the N terminus of a neighboring cyclophilin H molecule in a substrate-like manner, indicating the capacity of joint binding to a substrate and to U4/U6-60K. Free and complexed cyclophilin H have virtually identical conformations suggesting that the U4/U6-60K binding site is pre-shaped and the peptidyl-prolyl-cis/trans isomerase activity is unaffected by complex formation. The complex defines a novel protein-protein interaction mode for a cyclophilin, allowing cyclophilin H to mediate interactions between different proteins inside the spliceosome or to initiate from its binding platforms isomerization or chaperoning activities.

Amino Acid Sequence↗

PubMed2003

Molecular architecture of the multiprotein splicing factor SF3b.

The splicing factor SF3b is a multiprotein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoprotein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined the three-dimensional structure of the human SF3b complex by single-particle electron cryomicroscopy at a resolution of less than 10 angstroms, allowing identification of protein domains with known structural folds. The best fit of a modeled RNA-recognition motif indicates that the protein p14 is located in the central cavity of the complex. The 22 tandem helical repeats of the protein SF3b155 are located in the outer shell of the complex enclosing p14.

Amino Acid Motifs↗

PubMed2002

Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method.

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly.

Centrifugation, Density Gradient↗

PubMed2002

Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome.

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.

Catalysis↗

PubMed2002

Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins.

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.

Amino Acid Sequence↗

PubMed2002

Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD-box protein.

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.

DEAD-box RNA Helicases↗

PubMed2002

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.

Base Sequence↗

PubMed2002

The ribosomal translocase homologue Snu114p is involved in unwinding U4/U6 RNA during activation of the spliceosome.

Snu114p is a yeast U5 snRNP protein homologous to the ribosomal elongation factor EF-2. Snu114p exhibits the same domain structure as EF-2, including the G-domain, but with an additional N-terminal domain. To test whether Snu114p in the spliceosome is involved in rearranging RNA secondary structures (by analogy to EF-2 in the ribosome), we created conditionally lethal mutants. Deletion of this N-terminal domain (snu114 Delta N) leads to a temperature-sensitive phenotype at 37 degrees C and a pre-mRNA splicing defect in vivo. Heat treatment of snu114 Delta N extracts blocked splicing in vitro before the first step. The snu114 Delta N still associates with the tri-snRNP, and the stability of this particle is not significantly impaired by thermal inactivation. Heat treatment of snu114 Delta N extracts resulted in accumulation of arrested spliceosomes in which the U4 RNA was not efficiently released, and we show that U4 is still base paired with the U6 RNA. This suggests that Snu114p is involved, directly or indirectly, in the U4/U6 unwinding, an essential step towards spliceosome activation.

Alternative Splicing↗

PubMed2002

Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles.

The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.

Base Sequence↗

PubMed2002

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.

DNA Mutational Analysis↗

PubMed2002

The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro.

The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-beta binding (IBB) domain and a COOH-terminal m3G-cap--binding region that bears no structural similarity to known import adaptors like importin-alpha (impalpha). Here, we show that recombinant snurportin1 and importin-beta (impbeta) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impalpha-dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1-impbeta, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impbeta and a snurportin1 mutant containing an impalpha IBB domain (IBBimpalpha), nuclear U1 snRNP import was Ran dependent. Furthermore, beta-galactosidase (betaGal) containing a snurportin1 IBB domain, but not IBBimpalpha-betaGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impbeta with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.

Active Transport, Cell Nucleus↗

PubMed2002

A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles.

Much attention is currently being devoted to questions of protein and RNA tertiary structures and to the quaternary arrangement of the individual macromolecules in ribonucleoprotein (RNP) particles. In this article we describe two complementary strategies that allow the identification of RNA-protein contact sites in assembled, nonlabeled RNP particles after UV crosslinking. The first combines immunoprecipitation of UV-irradiated RNP particles under mildly denaturing conditions followed by primer-extension analysis of the crosslinked (and thus coprecipitated) RNA. The second involves the purification of crosslinked peptide-oligonucleotide from RNP particles and the subsequent analysis of the crosslinked peptide and RNA by Edman degradation and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), respectively. Although the first approach provides a rapid method for the exact identification of RNA-protein contact sites in purified nonlabeled RNP particles, the latter adds valuable information about potential RNA binding domains within proteins and, thus, about the arrangement of these proteins within the quaternary structures of complex RNP assemblies. Recently, we applied both these strategies successfully to native purified spliceosomal RNP. These methods may be generally applicable to the analysis of RNP complexes, especially as they avoid labeling and reconstitution, both of which risk introducing artifacts.

Binding Sites↗

PubMed2002

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions.

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.

Antibodies↗