Biochemistry and regulation of pre-mRNA splicing

During the past year, significant advances have been made in the field of pre-mRNA splicing. It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle. Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns. By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.     

Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C)

Enterotoxigenic Escherichia coli elaborate a peptide called heat-stable enterotoxin (ST), which binds to and activates the intestinal ST receptor (STaR). STaR, also known as guanylyl cyclase C (GC-C), is a member of the transmembrane guanylyl cyclase receptor family. The mRNA for STaR encodes an approximately 120 kDa protein with the N-terminal ligand binding domain on the cell surface. Ligand affinity cross-linking studies have previously demonstrated several species of potential ST binding proteins, ranging in size from approximately 50 to 160 kDa. Although these smaller forms of STaR (50-80 kDa) have been proposed to act in vivo as toxin binding proteins, their biogenesis and localization have not previously been examined. Using pulse labeling in vivo and synchronized translation in vitro, we demonstrate that these smaller forms represent incomplete translational products and are not formed through limited proteolysis of the full-length receptor, as had previously been believed. We determined, using fluorescence confocal microscopy and surface labeling, that only approximately 25% of cellular receptors are expressed at the surface, while the remaining population is retained within the endoplasmic reticulum. Only full-length receptor is found at the surface of the cell, indicating this to be the biologically active form of STaR responsible for interacting with the heat-stable enterotoxin and other luminal intestinal peptides. The large intracellular receptor population, and potential for function before translocation to the cell surface, may impact on how pharmacologic modulators of this clinically important receptor are designed.     

Gate-activated photoresponse in a graphene p-n junction

We study photodetection in graphene near a local electrostatic gate, which enables active control of the potential landscape and carrier polarity. We find that a strong photoresponse only appears when and where a p-n junction is formed, allowing on-off control of photodetection. Photocurrents generated near p-n junctions do not require biasing and can be realized using submicrometer gates. Locally modulated photoresponse enables a new range of applications for graphene-based photodetectors including, for example, pixilated infrared imaging with control of response on subwavelength dimensions.     

Public knowing of risk and children's independent mobility

Risk is major factor that influences parent decision-making about whether they let their children go places by themselves. However, there is a paucity of research about how risk is conceived. This paper presents research that identified parents’, local government general managers’, and regulatory document conceptions about different dimensions of risk, and how these may be influenced by worry and expert knowledge. These dimensions included views on the environment, children's competence, probability of adverse situations occurring, number and potential long-term impacts if adverse situations were encountered. The research also examined how the views of parents, local government general managers, and regulatory documents interacted to create a public knowing of risk that limits children's independent mobility (CIM). The results indicate that children had limited independent mobility. This was supported by narrow views of children's places and promotion of adult surveillance. Parents were more concerned about externally imposed situations representing deliberate harm by others rather than everyday type situations. Parents’ views were reflected by a variety of regulatory documents that positioned children as vulnerable to many adverse situations leading to potential longterm damage. While, general managers thought children should be permitted to use public space by themselves, they balanced their views with perceptions of parent and community tolerance for children in public space, and the regulatory environment in which they worked. Fieldwork was conducted in the western suburbs of Melbourne, Australia. It comprised a questionnaire completed by 160 parents, interviews with three general managers, and analysis of 237 regulatory documents from multiple levels of government.     

Hot carrier transport and photocurrent response in graphene

Strong electron-electron interactions in graphene are expected to result in multiple-excitation generation by the absorption of a single photon. We show that the impact of carrier multiplication on photocurrent response is enhanced by very inefficient electron cooling, resulting in an abundance of hot carriers. The hot-carrier-mediated energy transport dominates the photoresponse and manifests itself in quantum efficiencies that can exceed unity, as well as in a characteristic dependence of the photocurrent on gate voltages. The pattern of multiple photocurrent sign changes as a function of gate voltage provides a fingerprint of hot-carrier-dominated transport and carrier multiplication.     

A C. elegans mutant that lives twice as long as wild type

We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. Both genes also regulate formation of the dauer larva, a developmentally arrested larval form that is induced by crowding and starvation and is very long-lived. Our findings raise the possibility that the longevity of the dauer is not simply a consequence of its arrested growth, but instead results from a regulated lifespan extension mechanism that can be uncoupled from other aspects of dauer formation. daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.     

RNA binding activity of heterodimeric splicing factor U2AF: at least one RS domain is required for high-affinity binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3' splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3' splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF38) was complexed with the large-subunit (dU2AF50) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF50. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF65. Surprisingly, the RS domain on dU2AF38 was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF50DeltaRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50DeltaRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

A low noise SIS array receiver for radioastronomical applications in the 35–50 GHz band

Arrays of six superconducting tunnel junctions have been used in a heterodyne receiver over the frequency range 35–50 GHz. The mixer array and a 3.7–4.2 GHz parametric amplifier used as the if amplifier are immersed in liquid helium and operated at 2 K. The high if allows single sideband operation with a system noise temperature varying rather smoothly from 220 K at 35 GHz to 140 K at 50 GHz. Mixer noise temperatures between 11 and 21 K were measured over the band indicating that the use of arrays to enhance the dynamic range does not seriously affect the mixer noise performance in this frequency range. The receiver is used for radio astronomical observations in the Onsala 20 m telescope in Sweden.