Daniel Schümperli

Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon

Precise frameshift and nonsense mutations were introduced into the region preceding the galactokinase gene (galK) of Escherichia coli. These mutations after the position at which upstream translation terminates relative to the galK translation initiation signal. Constructions were characterized that allow ribosomes to stop selectively before, within or downstream from the galK initiation signal. The effects of these mutations on galK expression were monitored. Galactokinase levels are highest when upstream translation terminates within the galK initiation region. In contrast, when translation stops either upstream or down stream from the galK start site, galK expression is drastically reduced. These results demonstrate that the galK gene is translationally coupled to the gene immediately preceding galK in the gal operon (that is, galT), and that the coupling effect depends primarily on the position at which upstream translation terminates relative to the galK start site. Possible mechanisms and implications of this translational coupling phenomenon are discussed.

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein

The hairpin structure at the 3′ end of animal histone mRNAs controls histone RNA 3′ processing, nucleo‐cytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP‐encoding human cDNAs by RNA‐mediated three‐hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence‐ and structure‐specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S.cerevisiae was functional in histone pre‐mRNA processing, confirming that we have indeed identified the human HBP gene.

Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1‐like protein

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm‐like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm‐like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein

Abstract.The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3′ end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy.

Multilevel regulation of replication-dependent histone genes

Abstract The replication-dependent histone genes are regulated at the levels of transcription initiation, pre-mRNA 3′ processing and mRNA degradation. Characteristic sequence motifs at the 3′ ends of the genes appear to serve a dual function in both the processing and degradation modes of regulation.

Polydnavirus DNA of the braconid wasp Chelonus inanitus is integrated in the wasp's genome and excised only in later pupal and adult stages of the female

Many endoparasitic wasps inject, along with the egg, polydnavirus into their insect hosts, the virus being a prerequisite for successful parasitoid development. The genome of polydnaviruses consists of multiple circular dsDNA molecules of variable size. We show for a 12 kbp segment of the braconid Chelonus inanitus (CiV12) that it is integrated into the wasp genome. This is the first direct demonstration of integration for a bracovirus. PCR data indicated that the integrated form of CiV12 was present in all male and female stages investigated while the excised circular virus DNA only appeared in females after a specific stage in pupal-adult development. The data also indicated that after excision of virus DNA the genomic DNA was rejoined. This has not yet been reported for any polydnavirus. Sequence analyses in the junction regions revealed the presence of an imperfect consensus sequence of 15 nucleotides in CiV12, in each terminus of the integrated virus DNA and in the rejoined genomic DNA. Within these repeats two sequence types (ATA, TAC) were observed in the various virus clones and in the clones encompassing the rejoined genomic DNA; they corresponded to the sequence type in the right and left junction, respectively. To explain this, we propose a model of virus DNA replication in which the genomic DNA is folded to juxtapose the direct repeat of the left with that of the right junction; recombination at specific sites would then yield the two types of virus and rejoined genomic DNA.

POLYDNAVIRUS OF THE PARASITIC WASP CHELONUS INANITUS (BRACONIDAE) : CHARACTERIZATION, GENOME ORGANIZATION AND TIME POINT OF REPLICATION

Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.

Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation

In spinal muscular atrophy (SMA), the leading genetic cause of early childhood death, the survival motor neuron 1 gene (SMN1) is deleted or inactivated. The nearly identical SMN2 gene has a silent mutation that impairs the utilization of exon 7 and the production of functional protein. It has been hypothesized that therapies boosting SMN2 exon 7 inclusion might prevent or cure SMA. Exon 7 inclusion can be stimulated in cell culture by oligonucleotides or intracellularly expressed RNAs, but evidence for an in vivo improvement of SMA symptoms is lacking. Here, we unambiguously confirm the above hypothesis by showing that a bifunctional U7 snRNA that stimulates exon 7 inclusion, when introduced by germline transgenesis, can efficiently complement the most severe mouse SMA model. These results are significant for the development of a somatic SMA therapy, but may also provide new means to study pathophysiological aspects of this devastating disease.

Comparison of five poxvirus genomes by analysis with restriction endonucleases HindIII, BamI and EcoRI.

Summary The DNAs of rabbitpox, vaccinia, cowpox, ectromelia and fowl pox virus, all grown in the same host system, were cleaved with restriction endonucleases HindIII, BamI and EcoRI. The resulting digestion products were separated by electrophoresis on agarose slab gels. From the similarities and dissimilarities of the migration profiles obtained, it was concluded that vaccinia and rabbitpox virus are genetically the most closely related viruses investigated, although their DNA cleavage patterns are clearly distinguishable. Cowpox and ectromelia virus both show about the same degree of relatedness to each other as to rabbitpox and vaccinia virus. Fowl pox virus, which belongs to the genus Avipoxvirus, contains the largest genome examined (approx. 160 × 106 daltons) and the cleavage patterns of its DNA show no similarities with those of orthopoxvirus DNAs, thus indicating a very low degree of genetic relatedness. We believe that restriction analysis is a useful method for the identification and classification of poxvirus isolates.

RNA 3' processing regulates histone mRNA levels in a mammalian cell cycle mutant. A processing factor becomes limiting in G1-arrested cells

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.