Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.     

Golgi α1,4‐fucosyltransferase of Arabidopsis thaliana partially localizes at the nuclear envelope

We analyzed plant‐derived α1,4‐fucosyltransferase (FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc‐green fluorescent protein (GFP) or tomato LeFucTc‐GFP restored Lewis‐a formation in a fuctc mutant, confirming functionality in the trans‐Golgi. AtFucTc‐GFP partly accumulated at the nuclear envelope (NE) not observed for other homologs or truncated AtFucTc lacking the N‐terminus or catalytic domain. Analysis of At/LeFucTc‐GFP swap constructs with exchanged cytosolic, transmembrane and stalk (CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino‐acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N‐terminal GFP copies did, indicating localization at the inner nuclear membrane (INM). Tunicamycin treatment of AtFucTc‐GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N‐glycosylation. Yet neither expression in protoplasts of Arabidopsis N‐glycosylation mutants nor elimination of the N‐glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum (ER)‐to‐Golgi transport by co‐expression of Sar1(H74L) trapped tunicamycin‐released AtFucTc‐GFP in the ER, however, without NE localization. Since recovery after tunicamycin‐washout required de novo‐protein synthesis, our analyses suggest that AtFucTc localizes to the NE/INM due to interaction with an unknown (glyco)protein.      

Green‐lighting green fluorescent protein: Faster and more efficient folding by eliminating a cis–trans peptide isomerization event

Wild‐type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis–trans peptide bond isomerization. The only conserved cis‐peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X‐P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans‐peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.     

HOPS prevents the disassembly of trans‐SNARE complexes by Sec17p/Sec18p during membrane fusion

SNARE‐dependent membrane fusion requires the disassembly of cis‐SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans‐SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans‐SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans‐SNARE complex, like cis‐SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans‐, but not in the cis‐, configuration. This selective HOPS preservation of trans‐SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X‐100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans‐SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic.     

The influence of FKBP5 genotype on expression of FKBP5 and other glucocorticoid‐regulated genes,dependent on trauma exposure

The FK506 binding protein 51 (FKBP5), an intrinsic regulator of the glucocorticoid receptor, has been associated with pathological behaviors particularly in the context of childhood trauma (CT), via a putatively regulatory polymorphism, rs1360780. However, trans‐ and cis‐acting effects of this locus and its interaction with CT are incompletely understood. To study its effects on the expression of glucocorticoid‐regulated genes including FKBP5, we used lymphoblastoid cell lines (LCLs) derived from 16 CT‐exposed patients with greater than two substance dependence/suicidal behavior diagnoses (casesCT+) and 13 non‐CT‐exposed controls (controlsCT?). This study in LCLs measures long‐term trait‐like differences attributable to genotype or lasting epigenetic modification. Through analysis of differential allelic expression (DAE) using an FKBP5 3′‐UTR reporter single nucleotide polymorphism (SNP), rs3800373, that is in strong linkage disequilibrium with rs1360780, we confirmed that the rs1360780 risk allele (A) (or conceivably that of a linked SNP) leads to higher FKBP5 expression in controlsCT?. Intriguingly, casesCT+ did not show DAE, perhaps because of a genotype‐predicted difference in FKBP5 DNA methylation restricted to casesCT+. Furthermore, through correlation analyses on FKBP5 expression at baseline and after induction by dexamethasone, we observed that casesCT+ had lower induction of FKBP5 expression, indicating that overall they may have strong ultra‐short negative‐feedback. Only casesCT+ showed an effect of rs1360780 genotype on expression of FKBP5 and other glucocorticoid‐regulated genes. Together, these results confirm that the rs1360780 locus alters FKBP5 expression and further that in trans‐fashion this locus affects the expression of other glucocorticoid‐regulated genes after a glucocorticoid challenge. The CT exposure appears to be essential for trans‐effects of rs1360780 on glucocorticoid‐regulated genes.     

Access to Enantiomerically Pure cis‐ and trans‐β‐Phenylproline by High‐Performance Liquid Chromatography Resolution

The preparation of all four stereoisomers of the proline analog that bears a phenyl group attached to the β carbon either cis or trans to the carboxylic acid (cis‐ and trans‐β‐phenylproline, respectively) has been addressed. The methodology developed allows access to multigram quantities of the target amino acids in enantiomerically pure form and suitably protected for use in peptide synthesis. Racemic precursors of cis‐β‐phenylproline and trans‐β‐phenylproline were prepared from easily available starting materials and subjected to high‐performance liquid chromatography enantioseparation. Semipreparative columns (250 × 20 mm) containing chiral stationary phases based on amylose (Chiralpak IA) (Daicel‐Chiral Technologies Europe, Illkirch, France) or cellulose (Chiralpak IC) were used respectively for the resolution of the cis‐ and trans‐β‐phenylproline precursors. Chirality, 24:1082‐1091, 2012. © 2012 Wiley Periodicals, Inc.     

When sensing is gambling: An experimental system reveals how plasticity can generate tunable bet‐hedging strategies

Genotypes can persist in unpredictable environments by “hedging their bets” and producing diverse phenotypes. Theoretical studies have shown that the phenotypic variability needed for a bet‐hedging strategy can be generated by factors either inside or outside an organism. However, sensing the environment and bet hedging are frequently treated as distinct evolutionary strategies. Furthermore, nearly all empirical studies of the molecular underpinnings of bet‐hedging strategies to date have focused on internal sources of variability. We took a synthetic approach and constructed an experimental system where a phenotypic trade‐off is mediated by actively sensing a cue present in the environment. We show that active sensing can generate a diversified bet‐hedging strategy. Mutations affecting the norm of reaction to the cue alter the diversification strategy, indicating that bet hedging by active sensing is evolvable. Our results indicate that a broader class of biological systems should be considered as potential examples of bet‐hedging strategies, and that research into the structure of environmental variability is needed to distinguish bet‐hedging strategies from adaptive plasticity.     

Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Low expression and instability during isolation are major obstacles preventing adequate structure‐function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C‐terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C‐termini (C_in) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C‐termini (C_out) to C_in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C_out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein‐detergent complex was identified using an extended fluorescence‐detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure‐function studies. Five MPs were successfully cleaved from the GFP tag by site‐specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C_out topology, yielding sufficient protein suitable for structure‐function studies and are superior to expression and purification in the absence GFP fusion tagging.     

Reversible non‐genetic phenotypic heterogeneity in bacterial quorum sensing

Bacteria co‐ordinate their social behaviour in a density‐dependent manner by production of diffusible signal molecules by a process known as quorum sensing (QS). It is generally assumed that in homogenous environments and at high cell density, QS synchronizes cells in the population to perform collective social tasks in unison which maximize the benefit at the inclusive fitness of individuals. However, evolutionary theory predicts that maintaining phenotypic heterogeneity in performing social tasks is advantageous as it can serve as a bet‐hedging survival strategy. Using Pseudomonas syringae and Xanthomonas campestris as model organisms, which use two diverse classes of QS signals, we show that two distinct subpopulations of QS‐responsive and non‐responsive cells exist in the QS‐activated population. Addition of excess exogenous QS signal does not significantly alter the distribution of QS‐responsive and non‐responsive cells in the population. We further show that progeny of cells derived from these subpopulations also exhibited heterogeneous distribution patterns similar to their respective parental strains. Overall, these results support the model that bacteria maintain QS‐responsive and non‐responsive subpopulations at high cell densities in a bet‐hedging strategy to simultaneously perform functions that are both positively and negatively regulated by QS to improve their fitness in fluctuating environments.     

Variability in thermal and phototactic preferences in Drosophila may reflect an adaptive bet‐hedging strategy

Organisms use various strategies to cope with fluctuating environmental conditions. In diversified bet‐hedging, a single genotype exhibits phenotypic heterogeneity with the expectation that some individuals will survive transient selective pressures. To date, empirical evidence for bet‐hedging is scarce. Here, we observe that individual Drosophila melanogaster flies exhibit striking variation in light‐ and temperature‐preference behaviors. With a modeling approach that combines real world weather and climate data to simulate temperature preference‐dependent survival and reproduction, we find that a bet‐hedging strategy may underlie the observed interindividual behavioral diversity. Specifically, bet‐hedging outcompetes strategies in which individual thermal preferences are heritable. Animals employing bet‐hedging refrain from adapting to the coolness of spring with increased warm‐seeking that inevitably becomes counterproductive in the hot summer. This strategy is particularly valuable when mean seasonal temperatures are typical, or when there is considerable fluctuation in temperature within the season. The model predicts, and we experimentally verify, that the behaviors of individual flies are not heritable. Finally, we model the effects of historical weather data, climate change, and geographic seasonal variation on the optimal strategies underlying behavioral variation between individuals, characterizing the regimes in which bet‐hedging is advantageous.     

<Emphasis Type="Italic">Cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> photoisomerization properties of GFP chromophore analogs

The photoswitching behaviour of the green fluorescent protein (GFP) chromophore and its analogs opens up exciting horizons for the engineering and development of molecular devices for high sensitivity in vivo studies. In this work we present the synthesis and photophysical study of four GFP chromophore analogs belonging to butenolide and pyrrolinone classes. These chromophores possess an intriguing photoinduced cis–trans isomerization mechanism. Stereochemical structural assignment was unambiguously performed by 1D Nuclear Overhauser Effect NMR measurements. The spectroscopic properties of both cis and trans isomers were studied, and photoconversion quantum yield for cis–trans isomerization was assessed to be in the 0.1–0.4 range. Finally, the ³J_C,H coupling constant in the ¹³C–C=C–H motif was in excellent agreement with theoretical DFT calculations, thus providing a further confirmation of cis–trans photoisomerization of the structurally analog GFP chromophore.