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Biomedical subjects

R Lührmann

Publications and source records attributed to R Lührmann.

At least 127 records · Page 7Linked to original sources
PubMed1990

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.

Animals↗
PubMed1990

Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model.

The solution structure of human U1 snRNA was investigated by using base-specific chemical probes (dimethylsulfate, carbodiimide, diethylpyrocarbonate) and RNase V1. Chemical reagents were employed under various conditions of salt and temperature and allowed information at the Watson-Crick base-pairing positions to be obtained for 66% of the U1 snRNA bases. Double-stranded or stacked regions were examined with RNase V1. The dat gained from these experiments extend and support the previous 2D model for U1snRNA. However, to elucidate some aspects of the solution data that could not be accounted for by the secondary structure model, the information gathered from structure probing was used to provide the experimental basis required to construct and to test a tertiary structure model by computer graphics modeling. As a result, U1 snRNA is shown to adopt an asymmetrical X-shape that is formed by two helical domains, each one being generated by coaxial stacking of helices at the U1 snRNA cruciform. Chemical reactivities and model building show that a few nucleotides, previously proposed to be unpaired, can form A.G and U.U non Watson-Crick base-pairs, notably in stem-loop B. The structural model we propose for regions G12 to A124 integrates stereochemical constraints and is based both on solution structure data and sequence comparisons between U1 snRNAs.

Alkylating Agents↗
PubMed1990

Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants.

The interaction of the U1-specific proteins 70k, A and C with U1 snRNP was studied by depleting gradually U1 snRNPs of the U1-specific proteins by Mono-Q chromatography at elevated temperatures (20-37 degrees C). U1 snRNP species were obtained which were selectively depleted of either protein C, A, C and A, or of all three U1-specific proteins C, A and 70k while retaining the common proteins B' to G. These various types of U1 snRNP particles were used to study the differential accessibility of defined regions of U1 RNA towards nucleases V1 and S1 dependent on the U1 snRNP protein composition. The data indicate that in the U1 snRNP protein 70k interacts with stem/loop A and protein A with stem/loop B of U1 RNA. The presence or absence of protein C did not affect the nuclease digestion patterns of U1 RNA. Our results suggest further that the binding of protein A to the U1 snRNP particle should be independent of proteins 70k and C. Mouse cells contain two U1 RNA species, U1a and U1b, which differ in the structure of stem/loop B, with U1a exhibiting the same stem/loop B sequence as U1 RNA from HeLa cells. When we used Mono Q chromatography to investigate possible structural differences in the two types of U1 snRNPs, we observed that protein A was always preferentially lost from U1b snRNP as compared to U1a snRNPs. This indicates that one consequence of the structural difference between U1a and U1b is a lowering of the strength of binding of protein A to U1b snRNP. The possible functional significance of this finding is discussed with respect to the fact that U1b RNA is preferentially expressed in embryonal cells.

Animals↗
PubMed1990

U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site.

One of the functions of U1 small nuclear ribonucleoprotein (snRNP) in the splicing reaction of pre-mRNA molecules is the recognition of the 5' splice site. U1 snRNP proteins as well as base-pair interactions between U1 snRNA and the 5' splice site are important for the formation of the snRNP-pre-mRNA complex. To determine which proteins are needed for complex formation, the ability of U1 snRNPs gradually depleted of the U1-specific proteins C, A, and 70k to bind to an RNA molecule containing a 5' splice site sequence was studied in a nitrocellulose filter binding assay. The most significant effect was always observed when protein C was removed, either alone or together with other U1-specific proteins; the binding was reduced by 50 to 60%. Complementation of protein C-deficient U1 snRNPs with purified C protein restored their 5' splice site binding activity. These data suggest that protein C may potentiate the base-pair interaction between U1 RNA and the 5' splice site.

Animals↗
PubMed1990

Autoantibody production against the U small nuclear ribonucleoprotein particle proteins E, F and G in patients with connective tissue diseases.

The nucleoplasmic U small nuclear ribonucleoprotein particles (snRNP) have a set of seven proteins in common which are designated B', B, D, D', E, F and G. Patients suffering from rheumatoid autoimmune diseases such as systemic lupus erythematosus often develop autoantibodies against the proteins B', B, and D. Here we describe a sensitive immunoassay which allows the specific detection of autoantibodies reacting with the E, F or G snRNP proteins. We were able to identify several patient sera containing autoantibodies against one or more of these proteins. This demonstrates that all snRNP proteins described so far are potentially antigenic in systemic rheumatoid diseases. The characterization of the antibodies showed an immunological cross-reactivity between the snRNP protein G and the 70-kDa protein of U1 snRNP. Several sera contained autoantibodies which were specific for the F snRNP protein.

Antibody Specificity↗
PubMed1990

Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family.

We have studied by electron microscopy the structures of native small nuclear ribonucleoprotein (snRNP) particles U2 and U5 from HeLa cells. The structure of native U2 snRNP is characterized by a main body 8 nm in diameter with one additional domain about 4 nm long and 6 nm wide. Electron micrographs show that the 20S U5 snRNP, which contains at least seven U5-specific proteins in addition to the common proteins, has an elongated structure measuring 20-23 nm in length and 11-14 nm in width. Two main structural domains can be distinguished: a small head and a large elongated body about twice the size of the head. In addition to the head, the body of the 20S U5 snRNP possesses three short protuberances. The U2 and U5 core RNP particles--that is, of the snRNPs U2 and U5 without the snRNP-specific proteins, look much simpler and smaller under the electron microscope. They both are round in shape with a diameter of approximately 8 nm. With respect to their size, appearance, and fine structure, the U2 and U5 snRNP cores not only closely resemble each other but also share these properties with the core domain of U1 snRNP. We propose that the characteristic shape of each of the major snRNP species U1, U2, U4/U6, and U5 is determined by (i) a core domain containing the proteins that are common to all members of this family, which has the same shape for each member, and (ii) peripheral structures, which for snRNPs U1, U2, and U5 arise from the specific proteins, that give each of these snRNP species its characteristic shape.

Antibodies, Monoclonal↗
PubMed1989

Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein.

The process of nuclear pre-messenger RNA splicing is similar in Saccharomyces cerevisiae and metazoan cells in that the two-step mechanism is identical and the reaction occurs in a large ribonucleoprotein complex, the spliceosome. Little is known, however, about the degree of conservation of splicing factors other than of the small nuclear RNAs (snRNAs). Yeast counterparts of the metazoan spliceosomal snRNAs (U1, U2, U4, U5 and U6) have been identified but, with the exception of U6, the yeast snRNAs are larger and sequence similarity is limited to short regions. By using antibodies against the yeast PRP8 protein, a pre-mRNA splicing factor of relative molecular mass 280,000 (Mr280K) stably associated with U5 small nuclear ribonucleoproteins (snRNPs), we have now identified an immunologically related protein in HeLa cell nuclear extracts. The HeLa cell protein has an Mr greater than 200K and is associated with purified 20S U5 snRNPs. This is the first report of phylogenetic conservation between yeast and man of a protein splicing factor.

Autoantibodies↗
PubMed1989

Analysis of genomic clones of the murine U1RNA-associated 70-kDa protein reveals a high evolutionary conservation of the protein between human and mouse.

We have isolated and characterised two overlapping lambda EMBL3 clones carrying sequences of the gene for the murine U1RNA-associated 70-kDa protein. The two clones cover around 23 kb of the 70-kDa protein gene including its 3' end. Southern blot hybridisation revealed the existence of a single copy of the 70-kDa protein gene in the mouse genome. The 23-kb-long portion of the 70-kDa protein gene is divided into eight exons. While most of the exons are quite small and are widely scattered throughout the DNA sequence, the last one consists of about 830 bp and encodes 226 amino acids of the 70-kDa protein, including the C-terminus. The predicted amino acid sequence of the region of the 70-kDa protein encoded by the genomic clones reveals high conservation of structure when it is compared with the sequence of the human 70-kDa protein. Interestingly, all deletions, additions and substitutions are localised exclusively within the C-terminus of the protein, accounting for a 5'-3' polarity with respect to protein conservation. Moreover, the analysis of the genomic sequences predicts the existence of multiple subclasses of mRNAs that may arise by alternative pre-mRNA splicing. A 72-bp alternative exon harboring an in-frame termination codon was also found in the mouse 70-kDa gene and shows, surprisingly, 100% nucleotide identity to its human counterpart.

Amino Acid Sequence↗
PubMed1989

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.

Chromatography, Affinity↗
PubMed1989

Electron microscopy of U1 small nuclear ribonucleoprotein particles: shape of the particle and position of the 5' RNA terminus.

Small nuclear ribonucleoprotein particles U1 (U1 snRNPs) from HeLa cells were purified to homogeneity and examined in the electron microscope by negative staining with the double carbon film technique. A survey of several hundred views of the U1 snRNP particles revealed a main body about 8 nm in diameter with two characteristic protuberances 4-7 nm long and 3-4 nm wide. Anti-m3G antibodies were then used to define the site of the m3G base in the 5' cap of the U1 snRNA, close to the site where base-pairing occurs with the 5' splice sequence of pre-mRNA. The m3G base is located on the body of the U1 snRNP, close to one of the protuberances and approximately opposite the other.

Antibodies, Monoclonal↗
PubMed1989

Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.

We have established an in vitro complementation system that has allowed us to investigate the role of individual purified snRNPs in the splicing of pre-mRNA molecules. For the preparation of snRNP-depleted nuclear extracts we have first removed the majority of endogenous snRNPs from the nuclear extracts by one passage over an anti-m3G column and then degraded the remaining snRNPs with micrococcal nuclease. The mixture of snRNPs U1, U2, U4/U6 and U5, obtained by anti-m3G immuno-affinity chromatography, was functionally active and able to restore the splicing of snRNP-depleted nuclear extracts. Mono-Q chromatography was used for further fractionation of the snRNPs U1-U6. This produced three fractions that were highly enriched in snRNPs U1 and U2, U5 and U4/U6 respectively. Conditions were found where addition of the [U1, U2] and the U4/U6 snRNP fractions to the snRNP-depleted nuclear extracts gave rise to the formation of splice intermediates in the absence of any 3' cleavage/exon 1-exon 2 product formation. Only when purified 20S U5 snRNPs were added did both steps of the splicing reaction occur efficiently. Our data suggest that U5 snRNP is absolutely required for the second step of splicing and is needed further for efficient initiation of the splicing reaction. The requirement for U5 snRNPs for splicing was corroborated by glycerol gradient sedimentation analysis of the respective reconstituted pre-mRNP complexes. Stable and efficient formation of 50-60S spliceosomes was observed only in the presence of all snRNPs.

Centrifugation, Density Gradient↗