Small molecule control of pre-mRNA splicing

SR proteins regulate alternative splicing by binding to exonic sequences where, via an arginine/serine-rich splicing activation domain, they enhance the binding of the spliceosome to the adjacent splice sites. Here, a system is described in which a nontoxic derivative of the small molecule rapamycin is used to control pre-mRNA splicing in vitro. This involves the rapamycin-dependent recruitment of a splicing activation domain located on one protein to a second protein bound to the pre-mRNA. These results provide a new approach to explore for regulating gene expression in vivo with small molecules by controlling pre-mRNA splicing.     

Getting the message out

In a recent issue of Molecular Cell, Schwer (2008) demonstrates that, during the latest stage of the splicing reaction, the RNA-dependent helicase Prp22 is deposited upon the downstream exon, where it subsequently strips the spliced messenger RNA from the spliceosome.     

Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.     

Analysis of the human neurexin genes: alternative splicing and the generation of protein diversity

The neurexins are neuronal proteins that function as cell adhesion molecules during synaptogenesis and in intercellular signaling. Although mammalian genomes contain only three neurexin genes, thousands of neurexin isoforms may be expressed through the use of two alternative promoters and alternative splicing at up to five different positions in the pre-mRNA. To begin understanding how the expression of the neurexin genes is regulated, we have determined the complete nucleotide sequence of all three human neurexin genes: NRXN1, NRXN2, and NRXN3. Unexpectedly, two of these, NRXN1 ( approximately 1.1 Mb) and NRXN3 ( approximately 1.7 Mb), are among the largest known human genes. In addition, we have identified several conserved intronic sequence elements that may participate in the regulation of alternative splicing. The sequences of these genes provide insight into the mechanisms used to generate the diversity of neurexin protein isoforms and raise several interesting questions regarding the expression mechanism of large genes.     

Die Dauer der Dunkeladaptation beim Fliegenauge nach Belichtung mit heterochromatischen Blitzen

Zusammenfassung Versuche mit heterochromatischen Lichtblitzen von 2 msec Dauer zeigen, daß die Dauer der Dunkeladaptation beim Calliphora-ERG von der Reihenfolge der Farbblitze abhängt. Versuche mit 2 diskreten Blitzabständen von 40 msec und 2 sec ergaben unterschiedliche Wirkungsspektren. Bei 40 msec Blitzabstand wirkt UV-Strahlung verlängernd auf die Adaptationszeit, bei 2 sec Abstand verkürzend. Die aufgenommenen Wirkungsspektren sprechen für das Auftreten eines kurzlebigen, bei 490 nm absorbierenden Intermediärfarbstoffes während der Potentialbildung und für das Auftreten eines stark im UV-Bereich absorbierenden Folgefarbstoffes sofort nach der Potentialbildung.

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Dark-adaptation after heterochromatic flash-illumination in the compound eye of the blow-fly

Summary The time course of dark adaptation (increase in amplitude of the electroretinogram) in the fly Calliphora was tested by light flashes (2 msec) of various wavelengths. The results showed that the time course depends on the wavelength of the test flashes, and on the interval between them. Using white test light the time for completion of dark adaptation increases with the interval between flashes, up to an interval of 20 sec. With longer intervals dark adaptation proceeds slightly faster. In contrast, using ultra-violet (UV) test light, a short (40 msec) interval increases the duration of the dark adaptation, and a longer (2 sec) interval reduces the duration. The results suggest that an intermediate photopigment is formed during the generation of the receptor potential. The pigment, which absorbs maximally at 490 nm, exists for a short period only. Immediately after the receptor potential is set up, an UV absorbing secondary pigment is formed.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft, SFB Bionach.     

Effects of recent increases in salinity and nutrient concentrations on the microbialite community of Lake Clifton (Western Australia): are the thrombolites at risk?

There has been a strong research focus on optical properties in temperate estuaries but very much less in tropical estuaries. These properties comprise light and beam attenuation dominated by suspended particulate matter, Chl a, and CDOM. Spatially and temporally distributed data on optical properties in a tropical wet and dry estuary are compared and discussed in relation to those of temperate estuaries. Sampling in the Nha Phu estuary, Vietnam, consisted of five stations on a transect from head to mouth that was sampled four times during dry conditions and three times during wet conditions between May 2006 and April 2008. Methods comprised CTD, optical measurements, and water sampling for suspended matter, Chl a, and CDOM. Results showed high light attenuation—K _d(PAR)—in wet conditions and low in dry. K _d(PAR) was highest at the estuary head and lower in the outer part. Spatial and temporal variations in K _d(PAR) were in general dominated by variations in suspended particulate matter concentrations in both wet and dry conditions. Chl a concentrations were low and showed no strong variations between wet and dry conditions. CDOM absorption coefficients were higher in wet conditions with high values at the head and lower in the central part of the estuary. The depth of the photic zone was reduced by up to 50% during wet conditions. A residence time in the estuary of 5–6 days was derived from the rate of change of K _d(PAR) after a period of heavy rain and discharge of freshwater into the estuary. This complied with a residence time of four and a half days derived from a basic physical relation. Optical properties were in general comparable to temperate estuaries in dry conditions although Chl a concentrations were lower in Nha Phu. A second distinctive point, as compared to temperate estuaries, was the episodic character with days of strong rainfall followed by longer periods of dry weather. All sampling, both wet and dry, was carried out in the dry season which implies a less definitive perception of wet and dry seasons.