U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3′ end processing

The 3′ cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3′ end processing

The 3′ cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factor

Nuclear antisense effects in cyclophilin A pre‐mRNA splicing by oligonucleotides: a comparison of tricyclo‐DNA with LNA

The nuclear antisense properties of a series of tricyclo (tc)‐DNA oligonucleotide 9-15mers, targeted against the 3′ and 5′ splice sites of exon 4 of cyclophilin A (CyPA) pre‐mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA‐oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence‐ and dose‐dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc‐oligonucleotides was found to be superior to that of the LNA‐oligonucleotides in all cases by a factor of at least 4-5. A tc‐oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2 µM concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction in CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc‐15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc‐ and LNA‐ oligonucleotides. The obtained results confirm the power of tc‐DNA for nuclear antisense applications. Moreover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV‐

Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation. With dual-specificity U7 snRNAs targeting the 3′ and 5′ splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effec

Von der externen Hilfe zur Mobilisierung lokaler Ressourcen

Seit fünf Jahren hat sich das Abkommen von Monterrey über Entwicklungsfinanzierung bei den Akteuren der Entwicklungszusammenarbeit als Referenz durchgesetzt. Der „Konsens von Monterrey“, ein aus zwischenstaatlichen Verhandlungen hervorgegangener politischer Kompromiss, enthält in den Augen mancher Beobachter „wenig griffige Verpflichtungen und umschiffte strittige Fragen mit vagen Formulierungen“. Im Jahr 2008 wird in Doha eine Folgekonferenz zu den in Monterrey eingegangenen Verpflichtungen ..

Von der externen Hilfe zur Mobilisierung lokaler Ressourcen

Seit fünf Jahren hat sich das Abkommen von Monterrey über Entwicklungsfinanzierung bei den Akteuren der Entwicklungszusammenarbeit als Referenz durchgesetzt. Der „Konsens von Monterrey“, ein aus zwischenstaatlichen Verhandlungen hervorgegangener politischer Kompromiss, enthält in den Augen mancher Beobachter „wenig griffige Verpflichtungen und umschiffte strittige Fragen mit vagen Formulierungen“. Im Jahr 2008 wird in Doha eine Folgekonferenz zu den in Monterrey eingegangenen Verpflichtungen ..

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

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

U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping.

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs

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

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 Im68), participates in histone RNA 3′-end processing. CF Im68 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 Im68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF Im68, but inhibited by high amounts or by anti-CF Im68 antibody. Finally, immunoprecipitation of CF Im68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF Im subunit, CF Im25, does not appear to be involved in histone RNA processin

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

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 Im68), participates in histone RNA 3′-end processing. CF Im68 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 Im68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF Im68, but inhibited by high amounts or by anti-CF Im68 antibody. Finally, immunoprecipitation of CF Im68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF Im subunit, CF Im25, does not appear to be involved in histone RNA processing