155 results on '"Schümperli D"'
Search Results
2. The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein
- Author
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Schümperli, D. and Pillai, R. S.
- Published
- 2004
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3. U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping
- Author
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Brun, C., Suter, D., Pauli, C., Dunant, P., Lochmüller, H., Burgunder, J.-M., Schümperli, D., and Weis, J.
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- 2003
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4. mRNA 3' end processing and more--multiple functions of mammalian cleavage factor I-68
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Ruepp, MD, Schümperli, D, BARABINO, SILVIA MARIA LUISA, Ruepp, M, Schümperli, D, and Barabino, S
- Subjects
Histones ,Mammals ,mRNA Cleavage and Polyadenylation Factors ,RNA processing, RNA export, histone mRNA ,Animals ,Humans ,RNA, Messenger ,RNA 3' End Processing ,Models, Biological - Abstract
The formation of defined 3 ′ ends is an important step in the biogenesis of mRNAs. In eukaryotic cells, all mRNA 3 ′ ends are generated by endonucleolytic cleavage of primary transcripts in reactions that are essentially posttranscriptional. Nevertheless, 3 ′ end formation is tightly connected to transcription in vivo, and a link with mRNA export to the cytoplasm has been postulated. Here, we briefly review the current knowledge about the two types of mRNA 3 ′ end processing reactions, cleavage/polyadenylation and histone RNA processing. We then focus on factors shared between these two reactions. In particular, we discuss evidence for new functions of the mammalian cleavage factor I subunit CF I m68 in histone RNA 3 ′ processing and in the export of mature mRNAs from the nucleus to the cytoplasm. © 2010 John Wiley & Sons, Inc.
- Published
- 2011
5. Synthesis and cellular activity of stereochemically-pure 2′-O-(2-methoxyethyl)-phosphorothioate oligonucleotides
- Author
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Li, M., primary, Lightfoot, H. L., additional, Halloy, F., additional, Malinowska, A. L., additional, Berk, C., additional, Behera, A., additional, Schümperli, D., additional, and Hall, J., additional
- Published
- 2017
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6. Permeability changes of plasma and lysosomal membranes in HeLa cells infected with rabbit poxvirus
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Schümperli, D., Peterhans, E., and Wyler, R.
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- 1978
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7. Toward an assembly line for U7 snRNPs - Interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes
- Author
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Azzouz, T., Pillai, R., Däpp, C., Chari, A., Meister, G., Kambach, C., Fischer, U., and Schümperli, D.
- Abstract
The survival of motor neurons (SMN) complex mediates the assembly of small nuclear ribonucleoproteins (snRNPs) involved in splicing and histone RNA processing. A crucial step in this process is the binding of Sm proteins onto the SMN protein. For Sm B/B′, D1, and D3, efficient binding to SMN depends on symmetrical dimethyl arginine (sDMA) modifications of their RG-rich tails. This methylation is achieved by another entity, the PRMT5 complex. Its pICln subunit binds Sm proteins whereas the PRMT5 subunit catalyzes the methylation reaction. Here, we provide evidence that Lsm10 and Lsm11, which replace the Sm proteins D1 and D2 in the histone RNA processing U7 snRNPs, associate with pICln in vitro and in vivo without receiving sDMA modifications. This implies that the PRMT5 complex is involved in an early stage of U7 snRNP assembly and hence may have a second snRNP assembly function unrelated to sDMA modification. We also show that the binding of Lsm10 and Lsm11 to SMN is independent of any methylation activity. Furthermore, we present evidence for two separate binding sites in SMN for Sm/Lsm proteins. One recognizes Sm domains and the second one, the sDMA-modified RG-tails, which are present only in a subset of these proteins.
- Published
- 2005
8. T.O.5 Spinal muscular atrophy SMA: SMNs spatial requirement and therapies
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Burghes, A., primary, McGovern, V., additional, Porensky, P., additional, Duque, S., additional, Bevan, A., additional, Foust, K., additional, Odermatt, P., additional, Nlend, R., additional, Massoni-Laporte, A., additional, Mitrpant, C., additional, Wilton, S., additional, Schümperli, D., additional, and Kaspar, B., additional
- Published
- 2012
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9. Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes
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Marquis, J, primary, Kämpfer, S S, additional, Angehrn, L, additional, and Schümperli, D, additional
- Published
- 2008
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10. The low abundance of U7 snRNA is partly determined by its Sm binding site.
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Grimm, C., primary, Stefanovic, B., additional, and Schümperli, D., additional
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- 1993
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11. Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors.
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Melin, L., primary, Soldati, D., additional, Mital, R., additional, Streit, A., additional, and Schümperli, D., additional
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- 1992
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12. Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.
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Harris, M E, primary, Böhni, R, additional, Schneiderman, M H, additional, Ramamurthy, L, additional, Schümperli, D, additional, and Marzluff, W F, additional
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- 1991
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13. Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system.
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Martin, F, Michel, F, Zenklusen, D, Müller, B, and Schümperli, D
- Abstract
We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.
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- 2000
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14. Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA
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Soldati, D and Schümperli, D
- Abstract
Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.
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- 1988
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15. 3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors.
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Birchmeier, C, Schümperli, D, Sconzo, G, and Birnstiel, M L
- Abstract
In vitro-synthesized transcripts of the sea urchin histone H2A gene with 3' extensions are efficiently and rapidly processed to H2A mRNA with faithful 3' ends in Xenopus laevis oocyte nuclei. Processing requires the presence of a histone-specific dyad symmetry element and of H2A-proximal spacer sequences in the precursor RNA. In DNA injection experiments with a processing-deficient H2A mutant, the transcription products appear to terminate heterogeneously in the first 100-200 base pairs of the post-H2A spacer. Processing of synthetic H3 RNA precursors requires the prior injection of a 60-nucleotide RNA from sea urchin embryos that seems to be a component of a small nuclear ribonucleoprotein.
- Published
- 1984
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16. A signal regulating mouse histone H4 mRNA levels in a mammalian cell cycle mutant and sequences controlling RNA 3′ processing are both contained within the same 80‐bp fragment.
- Author
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Stauber, C., Lüscher, B., Eckner, R., Lötscher, E., and Schümperli, D.
- Abstract
Fragments from the 3′ end of a mouse histone H4 gene, when introduced into transcription units controlled by the SV40 early promoter, yield correctly processed RNA with histone‐specific 3′ ends, both in monkey and mouse cell lines. The processed RNA is regulated in parallel with endogenous H4 mRNAs in 21‐Tb cells, a temperature‐sensitive mouse mastocytoma cell cycle mutant that is specifically blocked in G1 phase at the non‐permissive temperature. Mutational analyses of the H4 gene fragment indicate that the minimal sequences for this regulation and for RNA 3′ processing are both contained within the same 80 bp. This fragment contains two histone‐specific, highly conserved sequence elements that are located at the 3′ end of histone mRNA and in the adjacent spacer region, respectively. Our data suggest that the observed cell cycle regulation is achieved either at RNA 3′ processing or at some later step involving the conserved 3′‐terminal sequence element of mature histone mRNA.
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- 1986
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17. Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA
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Wittek, R, Menna, A, Müller, H K, Schümperli, D, Boseley, P G, and Wyler, R
- Abstract
In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.
- Published
- 1978
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18. Affecting gene expression by altering the length and sequence of the 5' leader.
- Author
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Johansen, H, Schümperli, D, and Rosenberg, M
- Abstract
We have utilized a recombinant vector system designed to study gene control elements by fusing them to the Escherichia coli galactokinase gene (galK) to examine the effects of gene expression that result from introducing changes into the 5' noncoding leader region of the galK transcription unit. We varied the length of the 5' leader region and found no effect on galK expression, provided that the galK initiation codon remained the first AUG in the transcription unit. Using synthetic linkers, we then inserted specific sequences, each containing a single AUG codon at a defined position within the leader region. We found that the AUG inserts had widely different effects on galK expression and that the sequences surrounding the inserted AUG codons determined the magnitude of these effects. In addition, we placed these upstream inserted AUG codons into each of the three possible translation reading frames so that translation occurring in these frames terminates prior to, within, or downstream of the galK initiation codon, respectively. Single-nucleotide frameshift mutations were also introduced into one of these constructs to shift upstream translation into the other two possible reading frames. Depending upon where upstream translation stopped relative to the galK initiation codon, we observed consistently different effects on galK expression. Our results show that an upstream AUG that interferes with downstream translation initiation exerts its greatest effect when it translates out-of-frame through the downstream initiator into the gene. If translation is stopped upstream of or within the initiator, an unexpectedly high level of expression from the downstream AUG is maintained.
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- 1984
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19. Efficient expression of Escherichia coli galactokinase gene in mammalian cells.
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Schümperli, D, Howard, B H, and Rosenberg, M
- Abstract
The Escherichia coli galactokinase gene (galK) was inserted into a modified early region transcription unit of simian virus 40 (SV40) contained on a bacterial plasmid. Introduction of this pSVK vector into monkey, mouse, and hamster cell lines by transfection resulted in efficient expression of the bacterial galK gene. This expression was shown to be dependent upon fusion of the galK gene to the early promoter of SV40 and did not appear to require SV40 splice signals. Moreover, expression in these cells could be obtained either transiently, 24--72 hr after transfection, or continuously, after stable transformation. In particular, pSVK-dependent galK expression was obtained in a hamster cell line genetically deficient in galactokinase activity. Expression of the bacterial enzyme was shown to complement the galactosemic defect of these cells, thereby allowing their selective survival and growth on galactose as the only carbon source. The ability to readily assay, select for, and potentially select against galK expression from pSVK and its derivatives should prove extremely useful in studying eukaryotic gene regulatory signals.
- Published
- 1982
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20. The primary structure of the precursor of chicken mitochondrial aspartate aminotransferase. Cloning and sequence analysis of cDNA.
- Author
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Jaussi, R, Cotton, B, Juretić, N, Christen, P, and Schümperli, D
- Abstract
The mitochondrial isoenzyme of aspartate amino-transferase (mAspAT; subunit Mr 45,000) is synthesized on free polysomes in the cytosol as a precursor of higher Mr (pre-mAspAT; Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). We have isolated three overlapping cDNA clones that correspond almost to the full length of pre-mAspAT mRNA with 100 nucleotides at the 5‘ end missing. The mRNA is 2.1 kilobase pairs long and has a 3‘ noncoding region of 0.7 kilobase pairs. The cDNAs code for the 401 amino acid residues of mAspAT plus an NH2-terminal pre-piece. Deviations from the reported amino acid sequence were found at positions 154 and 202 where the cDNA specifies Gln instead of Glu. The pre-piece consists of 22 amino acid residues, among them 4 arginine and no acidic residues.
- Published
- 1985
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21. HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA
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Wittek, R, Menna, A, Schümperli, D, Stoffel, S, Müller, H K, and Wyler, R
- Abstract
The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.
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- 1977
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22. The structure of tandem regulatory regions in the deooperon of Escherichia coliK12
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Valentin‐Hansen, P., Aiba, H., and Schümperli, D.
- Abstract
The transcription initiation sites of the deooperon of Escherichia coliK12 have been determined in vitroand in vivo. Initiation of transcription occurs at two, tandemly arranged, homologous regions which are located 600 base pairs apart from each other. Evidence for a novel regulation mechanism of deogene expression involving the deoR repressor is presented.
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- 1982
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23. RNA 3′ processing regulates histone mRNA levels in a mammalian cell cycle mutant. A processing factor becomes limiting in G1‐arrested cells.
- Author
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Lüscher, B. and Schümperli, D.
- Abstract
Post‐transcriptional regulation of histone gene expression in a mouse mastocytoma cell cycle mutant (21‐Tb) depends largely on conserved DNA sequences that are essential for RNA 3′ processing. We have analyzed whether this regulation occurs at the level of RNA 3′ processing. We show, by RNase mapping, that nuclear H4 mRNA precursors, which are hardly detectable in total RNA from exponentially dividing cells, accumulate in G1‐arrested cells, i.e. when mature mRNAs are drastically reduced. Furthermore, we show that a heat‐labile component of the processing apparatus, recently identified in HeLa cell nuclear extracts, is limiting in extracts from G1‐arrested 21‐Tb cells. In contrast, this activity is in excess in extracts from exponentially dividing cells, whereas both extracts contain similar amounts of snRNPs of the Sm serotype. These fluctuations in the heat‐labile activity may generally contribute to proliferation or cell cycle dependent histone gene regulation.
- Published
- 1987
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24. Faithful cell-cycle regulation of a recombinant mouse histone H4 gene is controlled by sequences in the 3'-terminal part of the gene.
- Author
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Lüscher, B, Stauber, C, Schindler, R, and Schümperli, D
- Abstract
We have analyzed the expression of endogenous histone H4 genes and of a newly introduced H4 gene in 21-Tb cells, a mouse mastocytoma cell-cycle mutant. Endogenous H4 mRNAs were less abundant by a factor of 120-180 in G1-arrested than in exponentially multiplying cells. However, H4 transcription rates were only decreased by a factor of 3 under these conditions, as determined by in vitro elongation of nascent transcripts. This indicates that post-transcriptional control of histone mRNA levels is important, in accord with published data. We introduced a mouse H4 gene, modified by a 12-base-pair (bp) insertion in its coding sequence, into 21-Tb cells by DNA-mediated gene transfer. The levels of transcripts from this gene were regulated in parallel with those of the endogenous genes. Moreover, fusion of the simian virus 40 (SV40) early promoter to a 463-bp fragment containing the 3'-terminal half of the mouse H4 gene, including 230 bp of spacer sequences, led to the regulated expression of SV40/H4 fusion RNA. However, a small proportion of SV40-initiated transcripts were not processed to histone-specific 3' ends, but extended farther through the downstream Escherichia coli galactokinase gene to a SV40 polyadenylylation site. In contrast to the short SV40/H4 RNA, the levels of these longer transcripts were not reduced in G1-arrested cells. These results show that sequences in the 3'-terminal part of the H4 gene can regulate gene expression in the cell cycle, presumably at the post-transcriptional level, as long as they are not positioned much more distant from the terminus than normal.
- Published
- 1985
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25. The structure of tandem regulatory regions in the deo operon of Escherichia coli K12
- Author
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Valentin-Hansen, P., primary, Aiba, H., additional, and Schümperli, D., additional
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- 1982
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26. Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon
- Author
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Schümperli, D
- Published
- 1982
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27. Cell-cycle regulation of histone gene expression
- Author
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Schümperli, D
- Published
- 1986
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28. Mammalian pre-mRNA 3′ end processing factor CF Im68 functions in mRNA export
- Author
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Simona Paro, Silvia Vivarelli, Silvia M.L. Barabino, Daniel Schümperli, Stefano Cardinale, Marc-David Ruepp, Chiara Aringhieri, Aringhieri, C, Ruepp, M, Vivarelli, S, Cardinale, S, Paro, S, Schümperli, D, and Barabino, S
- Subjects
Nucleocytoplasmic Transport Proteins ,Transcription, Genetic ,Active Transport, Cell Nucleus ,Receptors, Cytoplasmic and Nuclear ,Karyopherins ,Biology ,Models, Biological ,RNA Transport ,NXF1 ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Polysome ,Centrifugation, Density Gradient ,RNA Precursors ,medicine ,Animals ,Humans ,Gene silencing ,Molecular Biology ,030304 developmental biology ,Ribonucleoprotein ,Cell Nucleus ,mRNA Cleavage and Polyadenylation Factors ,0303 health sciences ,Messenger RNA ,RNA processing, SR proteins, RNA export ,RNA-Binding Proteins ,Articles ,Cell Biology ,BIO/11 - BIOLOGIA MOLECOLARE ,Molecular biology ,Cell biology ,Protein Subunits ,Cell nucleus ,medicine.anatomical_structure ,Ribonucleoproteins ,Cytoplasm ,030220 oncology & carcinogenesis ,NIH 3T3 Cells ,RNA 3' End Processing ,Precursor mRNA ,Ribosomes ,HeLa Cells ,Protein Binding - Abstract
Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3′ end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3′ end processing complex, CF Im68, stimulates mRNA export. CF Im68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF Im68 may act as a novel adaptor for NXF1/TAP, we show that CF Im68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF Im68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3′ end processing factor CF Im68 in mRNA export.
- Published
- 2009
29. Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals.
- Author
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Halloy F, Brönnimann P, Hall J, and Schümperli D
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- Animals, Mass Spectrometry methods, Tissue Distribution, Oligonucleotides chemistry, Peptides chemistry
- Abstract
We describe methods to follow the fate of oligonucleotides after their injection into experimental animals. The quantitation in various tissues, blood or bone marrow cells is possible by chemical ligation PCR. This method works independently of chemical modifications of the oligonucleotide and/or its conjugations to lipid or peptide moieties. Moreover, metabolization intermediates can be detected by mass spectrometry. Together with a readout assay for the biochemical or physiological effects, which will differ, depending on the particular purpose of the oligonucleotide, these methods allow for a comprehensive understanding of oligonucleotide behavior in a living organism., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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30. Repurposing of glycine transport inhibitors for the treatment of erythropoietic protoporphyria.
- Author
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Halloy F, Iyer PS, Ghidini A, Lysenko V, Barman-Aksözen J, Grubenmann CP, Jucker J, Wildner-Verhey van Wijk N, Ruepp MD, Minder EI, Minder AE, Schneider-Yin X, Theocharides APA, Schümperli D, and Hall J
- Subjects
- Cells, Cultured, Erythrocytes drug effects, Erythrocytes metabolism, Glycine Plasma Membrane Transport Proteins metabolism, Humans, K562 Cells, Molecular Structure, Protoporphyria, Erythropoietic metabolism, Glycine Plasma Membrane Transport Proteins antagonists & inhibitors, Protoporphyria, Erythropoietic drug therapy, Protoporphyrins pharmacology
- Abstract
Erythropoietic protoporphyria (EPP) is a rare disease in which patients experience severe light sensitivity. It is caused by a deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis (HBS). The lack of FECH causes accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in patients' erythrocytes. Here, we explored an approach for the treatment of EPP by decreasing PPIX synthesis using small-molecule inhibitors directed to factors in the HBS pathway. We generated a FECH-knockout clone from K562 erythroleukemia cells, which accumulates PPIX and undergoes oxidative stress upon light exposure. We used these matched cell lines to screen a set of publicly available inhibitors of factors in the HBS pathway. Inhibitors of the glycine transporters GlyT1 and GlyT2 lowered levels of PPIX and markers of oxidative stress selectively in K562
11B4 cells, and in primary erythroid cultures from an EPP patient. Our findings open the door to investigation of glycine transport inhibitors for HBS disorders., Competing Interests: Declaration of interests J.B.A. is a founding member and vice president of the International Porphyria Patient Network, a not-for-profit patient organization. A.M. received funding from Clinuvel Pharmaceuticals for research on EPP., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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31. Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.
- Author
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Halloy F, Iyer PS, Ćwiek P, Ghidini A, Barman-Aksözen J, Wildner-Verhey van Wijk N, Theocharides APA, Minder EI, Schneider-Yin X, Schümperli D, and Hall J
- Subjects
- Albumins metabolism, Animals, Bone Marrow metabolism, COS Cells, Chlorocebus aethiops, Disease Models, Animal, Ferrochelatase metabolism, Humans, K562 Cells, Mice, Oligonucleotides blood, Oligonucleotides chemistry, Oligonucleotides pharmacokinetics, Polymorphism, Single Nucleotide, Protoporphyria, Erythropoietic genetics, Protoporphyria, Erythropoietic therapy, RNA Splice Sites, Tissue Distribution, Ferrochelatase genetics, Oligonucleotides administration & dosage, Protoporphyria, Erythropoietic metabolism, RNA Splicing
- Abstract
Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
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32. Delta-aminolevulinic acid synthase 2 expression in combination with iron as modifiers of disease severity in erythropoietic protoporphyria.
- Author
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Barman-Aksözen J, Halloy F, Iyer PS, Schümperli D, Minder AE, Hall J, Minder EI, and Schneider-Yin X
- Subjects
- Biosynthetic Pathways, Ferrochelatase genetics, Humans, Iron blood, Iron Regulatory Protein 2 metabolism, Iron-Regulatory Proteins metabolism, K562 Cells, Protoporphyria, Erythropoietic therapy, Protoporphyrins metabolism, 5-Aminolevulinate Synthetase genetics, 5-Aminolevulinate Synthetase metabolism, Iron metabolism, Protoporphyria, Erythropoietic enzymology
- Abstract
Deficiency in ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway, leads to an accumulation of protoporphyrin IX (PPIX) that causes a severely painful phototoxic reaction of the skin in patients with erythropoietic protoporphyria (EPP). Besides phototoxicity of the skin, EPP patients often present with symptoms of iron deficiency in form of a microcytic and hypochromic anemia with low serum iron and ferritin. In addition, elevated aminolevulinic acid synthase 2 (ALAS2) both at the mRNA and protein levels have been observed among EPP patients. ALAS is the first enzyme in the pathway and exists in two isoforms, whereby the isoform 2 (ALAS2) is expressed exclusively in erythropoiesis. The mRNA of ALAS2 contains an iron response element (IRE) at its 5'UTR. When iron is limited, iron response element binding protein 2 (IRP2) binds to the IRE of ALAS2 mRNA and suppresses its translation. In this study, we demonstrated that iron deprivation increased the amount of ALAS2 mRNA as well as the ratio of ALAS2 to FECH mRNAs in cultured erythroleukemic K562 cells. At the protein level, however, iron deprivation in the cell line caused reductions in both enzymes as shown by the Western blot analysis. A comparable increase in the ratio of ALAS2 to FECH mRNAs was also found in EPP patients indicating an imbalance in heme biosynthesis. As iron cannot be completely missing from an organism, we assume that in EPP patients, a certain amount of ALAS2 mRNA is translated despite a partial deficiency of FECH. The increase in ALAS2 enzyme contributes to the accumulation in PPIX in the patients. Targeted inhibition of ALAS2 could therefore be a treatment option for EPP., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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33. Positive cofactor 4 (PC4) contributes to the regulation of replication-dependent canonical histone gene expression.
- Author
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Brzek A, Cichocka M, Dolata J, Juzwa W, Schümperli D, and Raczynska KD
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- Cell Cycle, Cleavage Stimulation Factor metabolism, Gene Expression Regulation, HEK293 Cells, HeLa Cells, Humans, RNA 3' End Processing, Ribonucleoprotein, U7 Small Nuclear metabolism, DNA Replication, DNA-Binding Proteins metabolism, Histones genetics, RNA Polymerase II metabolism, Transcription Factors metabolism
- Abstract
Background: Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this increment is achieved by both enhanced transcription and 3' end processing. In this paper, we described positive cofactor 4 (PC4) as a protein that contributes to the regulation of replication-dependent histone gene expression., Results: We showed that PC4 influences RNA polymerase II recruitment to histone gene loci in a cell cycle-dependent manner. The most important effect was observed in S phase where PC4 knockdown leads to the elevated level of RNA polymerase II on histone genes, which corresponds to the increased total level of those gene transcripts. The opposite effect was caused by PC4 overexpression. Moreover, we found that PC4 has a negative effect on the unique 3' end processing of histone pre-mRNAs that can be based on the interaction of PC4 with U7 snRNP and CstF64. Interestingly, this effect does not depend on the cell cycle., Conclusions: We conclude that PC4 might repress RNA polymerase II recruitment and transcription of replication-dependent histone genes in order to maintain the very delicate balance between histone gene expression and DNA synthesis. It guards the cell from excess of histones in S phase. Moreover, PC4 might promote the interaction of cleavage and polyadenylation complex with histone pre-mRNAs, that might impede with the recruitment of histone cleavage complex. This in turn decreases the 3' end processing efficiency of histone gene transcripts.
- Published
- 2018
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34. Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice.
- Author
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Barman-Aksözen J, C Wiek P, Bansode VB, Koentgen F, Trüb J, Pelczar P, Cinelli P, Schneider-Yin X, Schümperli D, and Minder EI
- Subjects
- Alleles, Alternative Splicing genetics, Animals, Base Sequence, Blood Cells pathology, Breeding, Disease Models, Animal, Exons genetics, Genotype, Homologous Recombination genetics, Humans, Light, Liver pathology, Mice, Inbred C57BL, Mice, Transgenic, Organ Size, Protoporphyria, Erythropoietic blood, Protoporphyria, Erythropoietic pathology, RNA, Messenger genetics, RNA, Messenger metabolism, Skin pathology, Skin radiation effects, Ferrochelatase genetics, Mutation genetics, Protoporphyria, Erythropoietic enzymology, Protoporphyria, Erythropoietic genetics
- Abstract
Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3' splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing., (© 2017. Published by The Company of Biologists Ltd.)
- Published
- 2017
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35. Somatic Therapy of a Mouse SMA Model with a U7 snRNA Gene Correcting SMN2 Splicing.
- Author
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Odermatt P, Trüb J, Furrer L, Fricker R, Marti A, and Schümperli D
- Subjects
- Animals, Disease Models, Animal, Genetic Vectors administration & dosage, Liver metabolism, Mice, Mice, Transgenic, Muscular Atrophy, Spinal genetics, Myocardium metabolism, RNA Splicing, RNA, Small Nuclear pharmacology, Adenoviridae genetics, Genetic Therapy methods, Muscular Atrophy, Spinal therapy, RNA, Small Nuclear administration & dosage, Survival of Motor Neuron 2 Protein genetics
- Abstract
Spinal Muscular Atrophy is due to the loss of SMN1 gene function. The duplicate gene SMN2 produces some, but not enough, SMN protein because most transcripts lack exon 7. Thus, promoting the inclusion of this exon is a therapeutic option. We show that a somatic gene therapy using the gene for a modified U7 RNA which stimulates this splicing has a profound and persistent therapeutic effect on the phenotype of a severe Spinal Muscular Atrophy mouse model. To this end, the U7 gene and vector and the production of pure, highly concentrated self-complementary (sc) adenovirus-associated virus 9 vector particles were optimized. Introduction of the functional vector into motoneurons of newborn Spinal Muscular Atrophy mice by intracerebroventricular injection led to a highly significant, dose-dependent increase in life span and improvement of muscle functions. Besides the central nervous system, the therapeutic U7 RNA was expressed in the heart and liver which may additionally have contributed to the observed therapeutic efficacy. This approach provides an additional therapeutic option for Spinal Muscular Atrophy and could also be adapted to treat other diseases of the central nervous system with regulatory small RNA genes.
- Published
- 2016
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- View/download PDF
36. Cycling in the nucleus: regulation of RNA 3' processing and nuclear organization of replication-dependent histone genes.
- Author
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Romeo V and Schümperli D
- Subjects
- Animals, Cell Cycle, Cell Cycle Checkpoints, Cell Nucleus metabolism, DNA Replication, Gene Expression, Humans, Polyadenylation, Histones genetics, RNA Precursors metabolism, RNA Processing, Post-Transcriptional, RNA, Messenger metabolism
- Abstract
The histones which pack new DNA during the S phase of animal cells are made from mRNAs that are cleaved at their 3' end but not polyadenylated. Some of the factors used in this reaction are unique to it while others are shared with the polyadenylation process that generates all other mRNAs. Recent work has begun to shed light on how the cell manages the assignment of these common components to the two 3' processing systems, and how it achieves their cell cycle-regulation and recruitment to the histone pre-mRNA. Moreover, recent and older findings reveal multiple connections between the nuclear organization of histone genes, their transcription and 3' end processing as well as the control of cell proliferation., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
37. Rudolf Weber (1922-2015): a driving force in the transition of developmental biology into a molecular and cellular science.
- Author
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Schümperli D
- Subjects
- Animals, Cell Biology history, History, 20th Century, History, 21st Century, Models, Animal, Molecular Biology history, Xenopus, Developmental Biology history
- Published
- 2016
- Full Text
- View/download PDF
38. Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.
- Author
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Neve A, Trüb J, Saxena S, and Schümperli D
- Subjects
- Animals, Apoptosis Inducing Factor metabolism, Diaphragm metabolism, Diaphragm physiopathology, Disease Models, Animal, Intracellular Signaling Peptides and Proteins metabolism, LIM Domain Proteins metabolism, Mice, Muscle Proteins metabolism, Muscular Atrophy, Spinal physiopathology, Neuromuscular Junction metabolism, Proteomics, Schwann Cells metabolism, Survival of Motor Neuron 1 Protein genetics, Synaptic Vesicles metabolism, Diaphragm innervation, Motor Neurons metabolism, Muscular Atrophy, Spinal metabolism, Survival of Motor Neuron 1 Protein metabolism
- Abstract
Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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- View/download PDF
39. FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors.
- Author
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Raczynska KD, Ruepp MD, Brzek A, Reber S, Romeo V, Rindlisbacher B, Heller M, Szweykowska-Kulinska Z, Jarmolowski A, and Schümperli D
- Subjects
- Cell Cycle, Cell Cycle Proteins metabolism, HEK293 Cells, HeLa Cells, Heterogeneous-Nuclear Ribonucleoproteins metabolism, Histones biosynthesis, Humans, Nuclear Proteins metabolism, Promoter Regions, Genetic, RNA, Small Nuclear metabolism, Transcription Factors metabolism, DNA Replication, Gene Expression Regulation, Histones genetics, RNA-Binding Protein FUS metabolism, Ribonucleoprotein, U7 Small Nuclear metabolism, Transcription, Genetic
- Abstract
Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2015
- Full Text
- View/download PDF
40. A large animal model of spinal muscular atrophy and correction of phenotype.
- Author
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Duque SI, Arnold WD, Odermatt P, Li X, Porensky PN, Schmelzer L, Meyer K, Kolb SJ, Schümperli D, Kaspar BK, and Burghes AH
- Subjects
- Animals, Biomarkers, Dependovirus genetics, Electromyography, Genetic Vectors therapeutic use, Humans, Motor Neurons pathology, Muscular Atrophy, Spinal etiology, Muscular Atrophy, Spinal pathology, Muscular Atrophy, Spinal physiopathology, Phenotype, RNA, Small Interfering therapeutic use, SMN Complex Proteins genetics, Swine, Disease Models, Animal, Genetic Therapy methods, Motor Neurons metabolism, Muscular Atrophy, Spinal therapy, SMN Complex Proteins metabolism
- Abstract
Objectives: Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers., Methods: Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed., Results: Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology., Interpretation: High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset., (© 2014 American Neurological Association.)
- Published
- 2015
- Full Text
- View/download PDF
41. CstF64: cell cycle regulation and functional role in 3' end processing of replication-dependent histone mRNAs.
- Author
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Romeo V, Griesbach E, and Schümperli D
- Subjects
- Apoptosis Regulatory Proteins genetics, Calcium-Binding Proteins genetics, Cell Line, Tumor, Cell Proliferation, Cleavage Stimulation Factor, HEK293 Cells, HeLa Cells, Humans, RNA Precursors genetics, RNA-Binding Proteins genetics, Ribonucleoprotein, U7 Small Nuclear genetics, S Phase genetics, Cleavage And Polyadenylation Specificity Factor genetics, Histones genetics, Nuclear Proteins genetics, RNA 3' End Processing genetics, RNA-Binding Proteins physiology
- Abstract
The 3' end processing of animal replication-dependent histone mRNAs is activated during G1/S-phase transition. The processing site is recognized by stem-loop binding protein and the U7 snRNP, but cleavage additionally requires a heat-labile factor (HLF), composed of cleavage/polyadenylation specificity factor, symplekin, and cleavage stimulation factor 64 (CstF64). Although HLF has been shown to be cell cycle regulated, the mechanism of this regulation is unknown. Here we show that levels of CstF64 increase toward the S phase and its depletion affects histone RNA processing, S-phase progression, and cell proliferation. Moreover, analyses of the interactions between CstF64, symplekin, and the U7 snRNP-associated proteins FLASH and Lsm11 indicate that CstF64 is important for recruiting HLF to histone precursor mRNA (pre-mRNA)-resident proteins. Thus, CstF64 is central to the function of HLF and appears to be at least partly responsible for its cell cycle regulation. Additionally, we show that misprocessed histone transcripts generated upon CstF64 depletion mainly accumulate in the nucleus, where they are targets of the exosome machinery, while a small cytoplasmic fraction is partly associated with polysomes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
- Full Text
- View/download PDF
42. The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.
- Author
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Voigt T, Neve A, and Schümperli D
- Subjects
- Animals, Diaphragm ultrastructure, Disease Models, Animal, Female, Intercostal Muscles ultrastructure, Mice, Disease Progression, Muscle Development, Muscle, Skeletal ultrastructure, Muscular Atrophy, Spinal pathology, Neuromuscular Junction ultrastructure
- Abstract
Aims: As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA., Methods: Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice., Results: During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes., Conclusion: SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis., (© 2013 British Neuropathological Society.)
- Published
- 2014
- Full Text
- View/download PDF
43. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.
- Author
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Huo Q, Kayikci M, Odermatt P, Meyer K, Michels O, Saxena S, Ule J, and Schümperli D
- Subjects
- Animals, Blotting, Western, Cell Line, Tumor, Cells, Cultured, Humans, Introns genetics, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Knockout, Mice, Transgenic, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal metabolism, Muscular Atrophy, Spinal pathology, Neuroblastoma genetics, Neuroblastoma metabolism, Neuroblastoma pathology, RNA Interference, RNA Splicing Factors, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, SMN Complex Proteins metabolism, Septins genetics, Septins metabolism, Gene Expression Regulation, Motor Neurons metabolism, RNA Splicing, SMN Complex Proteins genetics
- Abstract
Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.
- Published
- 2014
- Full Text
- View/download PDF
44. Antisense genes to induce exon inclusion.
- Author
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Nlend RN and Schümperli D
- Subjects
- Animals, Base Sequence, Blotting, Western methods, Genetic Therapy methods, Lentivirus genetics, Mice, Molecular Sequence Data, Plasmids genetics, Polymerase Chain Reaction methods, RNA genetics, RNA isolation & purification, Survival of Motor Neuron 2 Protein genetics, Transfection, Antisense Elements (Genetics) genetics, Cloning, Molecular methods, Exons, RNA, Small Nuclear genetics
- Abstract
Many inherited diseases are associated with changed splicing patterns, and alternative splicing influences several biological processes as well as the replication of certain viral pathogens. For this reason, there is a broad interest in modulating individual splicing events for therapeutic purposes. Based on the small nuclear RNA (snRNA) U7, we have developed expression vectors for short antisense RNAs that accumulate in the cell nucleus where splicing occurs and that can very specifically modulate the splicing of individual exons. More specifically, in the context of the fatal neuromuscular disorder Spinal Muscular Atrophy (SMA), we have shown that U7 snRNA constructs can restore the inclusion of exon 7 in the SMN2 gene and thereby alleviate or even fully cure disease symptoms in a severe mouse model for SMA. Here we describe more generally procedures to produce U7 constructs to induce exon inclusion and to test their efficiency in cell culture experiments at the level of RNA as well as protein. The analytical methods comprise reverse transcription (RT-)PCR to detect the splicing changes, quantitative real-time RT-PCR to measure U7 snRNA expression levels and western blot and immunofluorescence methods to detect a restoration of protein expression. Additionally, we indicate how U7 cassettes can be introduced into gene transfer vectors for in vivo experiments in animal models or to transduce cell systems that are not readily amenable to DNA transfection.
- Published
- 2012
- Full Text
- View/download PDF
45. Interactions of CstF-64, CstF-77, and symplekin: implications on localisation and function.
- Author
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Ruepp MD, Schweingruber C, Kleinschmidt N, and Schümperli D
- Subjects
- Blotting, Far-Western, Cleavage Stimulation Factor genetics, Fluorescent Antibody Technique, Gene Expression, HeLa Cells, Histones genetics, Histones metabolism, Humans, Nuclear Proteins chemistry, Nuclear Proteins genetics, Polyadenylation, Protein Binding, Protein Processing, Post-Translational, RNA 3' End Processing, RNA 3' Polyadenylation Signals, RNA Precursors genetics, RNA Precursors metabolism, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction, mRNA Cleavage and Polyadenylation Factors genetics, Cleavage Stimulation Factor chemistry, Cleavage Stimulation Factor metabolism, Nuclear Proteins metabolism, mRNA Cleavage and Polyadenylation Factors metabolism
- Abstract
Cleavage/polyadenylation of mRNAs and 3' processing of replication-dependent histone transcripts are both mediated by large complexes that share several protein components. Functional studies of these shared proteins are complicated by the cooperative binding of the individual subunits. For CstF-64, an additional difficulty is that symplekin and CstF-77 bind mutually exclusively to its hinge domain. Here we have identified CstF-64 and symplekin mutants that allowed us to distinguish between these interactions and to elucidate the role of CstF-64 in the two processing reactions. The interaction of CstF-64 with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation. In contrast, the nuclear accumulation of CstF-64 depends on its binding to CstF-77 and not to symplekin. Moreover, the CstF-64 paralogue CstF-64Tau can compensate for the loss of CstF-64. As CstF-64Tau has a lower affinity for CstF-77 than CstF-64 and is relatively unstable, it is the minor form. However, it may become up-regulated when the CstF-64 level decreases, which has biological implications for spermatogenesis and probably also for other regulatory events. Thus, the interactions between CstF-64/CstF-64Tau and CstF-77 are important for the maintenance of stoichiometric nuclear levels of the CstF complex components and for their intracellular localization, stability, and function.
- Published
- 2011
- Full Text
- View/download PDF
46. The 68 kDa subunit of mammalian cleavage factor I interacts with the U7 small nuclear ribonucleoprotein and participates in 3'-end processing of animal histone mRNAs.
- Author
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Ruepp MD, Vivarelli S, Pillai RS, Kleinschmidt N, Azzouz TN, Barabino SM, and Schümperli D
- Subjects
- Animals, Binding Sites, Cell Line, Histones metabolism, Humans, Mice, Mutation, Protein Subunits chemistry, Protein Subunits metabolism, RNA Precursors metabolism, RNA, Messenger metabolism, RNA-Binding Proteins, Ribonucleoproteins, Small Nuclear chemistry, Ribonucleoproteins, Small Nuclear genetics, mRNA Cleavage and Polyadenylation Factors chemistry, Histones genetics, RNA 3' End Processing, Ribonucleoprotein, U7 Small Nuclear metabolism, Ribonucleoproteins, Small Nuclear metabolism, mRNA Cleavage and Polyadenylation Factors metabolism
- Abstract
Metazoan replication-dependent histone pre-mRNAs undergo a unique 3'-cleavage reaction which does not result in mRNA polyadenylation. Although the cleavage site is defined by histone-specific factors (hairpin binding protein, a 100-kDa zinc-finger protein and the U7 snRNP), a large complex consisting of cleavage/polyadenylation specificity factor, two subunits of cleavage stimulation factor and symplekin acts as the effector of RNA cleavage. Here, we report that yet another protein involved in cleavage/polyadenylation, mammalian cleavage factor I 68-kDa subunit (CF I(m)68), participates in histone RNA 3'-end processing. CF I(m)68 was found in a highly purified U7 snRNP preparation. Its interaction with the U7 snRNP depends on the N-terminus of the U7 snRNP protein Lsm11, known to be important for histone RNA processing. In vivo, both depletion and overexpression of CF I(m)68 cause significant decreases in processing efficiency. In vitro 3'-end processing is slightly stimulated by the addition of low amounts of CF I(m)68, but inhibited by high amounts or by anti-CF I(m)68 antibody. Finally, immunoprecipitation of CF I(m)68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF I(m) subunit, CF I(m)25, does not appear to be involved in histone RNA processing.
- Published
- 2010
- Full Text
- View/download PDF
47. Ultrastructural changes in diaphragm neuromuscular junctions in a severe mouse model for Spinal Muscular Atrophy and their prevention by bifunctional U7 snRNA correcting SMN2 splicing.
- Author
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Voigt T, Meyer K, Baum O, and Schümperli D
- Subjects
- Animals, Diaphragm pathology, Disease Models, Animal, Mice, Mice, Transgenic, Microscopy, Electron, Transmission, Mitochondria diagnostic imaging, Mitochondria pathology, Motor Neurons pathology, Motor Neurons ultrastructure, Muscle, Skeletal diagnostic imaging, Muscle, Skeletal pathology, Muscular Atrophy, Spinal genetics, Neuromuscular Junction genetics, Neuromuscular Junction pathology, Reverse Transcriptase Polymerase Chain Reaction, Survival of Motor Neuron 2 Protein genetics, Ultrasonography, Diaphragm ultrastructure, Muscular Atrophy, Spinal pathology, Neuromuscular Junction ultrastructure
- Abstract
In Spinal Muscular Atrophy (SMA), the SMN1 gene is deleted or inactivated. Because of a splicing problem, the second copy gene, SMN2, generates insufficient amounts of functional SMN protein, leading to the death of spinal cord motoneurons. For a "severe" mouse SMA model (Smn -/-, hSMN2 +/+; with affected pups dying at 5-7 days), which most closely mimicks the genetic set-up in human SMA patients, we characterise SMA-related ultrastructural changes in neuromuscular junctions (NMJs) of two striated muscles with discrete functions. In the diaphragm, but not the soleus muscle of 4-days old SMA mice, mitochondria on both sides of the NMJs degenerate, and perisynaptic Schwann cells as well as endoneurial fibroblasts show striking changes in morphology. Importantly, NMJs of SMA mice in which a modified U7 snRNA corrects SMN2 splicing and delays or prevents SMA symptoms are normal. This ultrastructural study reveals novel features of NMJ alterations - in particular the involvement of perisynaptic Schwann cells - that may be relevant for human SMA pathogenesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
48. Repair of pre-mRNA splicing: prospects for a therapy for spinal muscular atrophy.
- Author
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Nlend Nlend R, Meyer K, and Schümperli D
- Subjects
- Animals, Exons, Humans, Mice, Models, Animal, Survival of Motor Neuron 2 Protein genetics, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal therapy, RNA Splicing
- Abstract
Recent analyses of complete genomes have revealed that alternative splicing became more prevalent and important during eukaryotic evolution. Alternative splicing augments the protein repertoire--particularly that of the human genome--and plays an important role in the development and function of differentiated cell types. However, splicing is also extremely vulnerable, and defects in the proper recognition of splicing signals can give rise to a variety of diseases. In this review, we discuss splicing correction therapies, by using the inherited disease Spinal Muscular Atrophy (SMA) as an example. This lethal early childhood disorder is caused by deletions or other severe mutations of SMN1, a gene coding for the essential survival of motoneurons protein. A second gene copy present in humans and few non-human primates, SMN2, can only partly compensate for the defect because of a single nucleotide change in exon 7 that causes this exon to be skipped in the majority of mRNAs. Thus SMN2 is a prime therapeutic target for SMA. In recent years, several strategies based on small molecule drugs, antisense oligonucleotides or in vivo expressed RNAs have been developed that allow a correction of SMN2 splicing. For some of these, a therapeutic benefit has been demonstrated in mouse models for SMA. This means that clinical trials of such splicing therapies for SMA may become possible in the near future.
- Published
- 2010
- Full Text
- View/download PDF
49. Editorial on alternative splicing and disease.
- Author
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Barta A and Schümperli D
- Subjects
- Humans, RNA Splice Sites, Alternative Splicing, Disease genetics
- Published
- 2010
- Full Text
- View/download PDF
50. Improving gene silencing of siRNAs via tricyclo-DNA modification.
- Author
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Ittig D, Luisier S, Weiler J, Schümperli D, and Leumann CJ
- Abstract
Small interfering RNAs (siRNAs) can be exploited for the selective silencing of disease-related genes via the RNA interference (RNAi) machinery and therefore raise hope for future therapeutic applications. Especially chemically modified siRNAs are of interest as they are expected to convert lead siRNA sequences into effective drugs. To study the potential of tricyclo-DNA (tc-DNA) in this context we systematically incorporated tc-DNA units at various positions in a siRNA duplex targeted to the EGFP gene that was expressed in HeLa cells. Silencing activity was measured by FACS, mRNA levels were determined by RT-PCR and the biostability of the modifed siRNAs was determined in human serum. We found that modifications in the 3'-overhangs in both the sense and antisense strands were compatible with the RNAi machinery leading to similar activities compared to wild-type (wt) siRNA. Additional modifications at the 3'-end, the 5'-end and in the center of the sense (passenger) strand were also well tolerated and did not compromise activity. Extensive modifications of the 3'- and the 5'-end in the antisense (guide) strand, however, abolished RNAi activity. Interestingly, modifications in the center of the duplex on both strands, corresponding to the position of the cleavage site by AGO2, increased efficacy relative to wt by a factor of 4 at the lowest concentrations (2 nM) investigated. In all cases, reduction of EGFP fluorescence was accompanied with a reduction of the EGFP mRNA level. Serum stability analysis further showed that 3'-overhang modifications only moderately increased stability while more extensive substitution by tc-DNA residues significantly enhanced biostability.
- Published
- 2010
- Full Text
- View/download PDF
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