Cotranslational microRNA mediated messenger RNA destabilization.

MicroRNAs are small (22 nucleotide) regulatory molecules that play important roles in a wide variety of biological processes. These RNAs, which bind to targeted mRNAs via limited base pairing interactions, act to reduce protein production from those mRNAs. Considerable evidence indicates that miRNAs destabilize targeted mRNAs by recruiting enzymes that function in normal mRNA decay and mRNA degradation is widely thought to occur when mRNAs are in a ribosome free state. Nevertheless, when examined, miRNA targeted mRNAs are invariably found to be polysome associated; observations that appear to be at face value incompatible with a simple decay model. Here, we provide evidence that turnover of miRNA-targeted mRNAs occurs while they are being translated. Cotranslational mRNA degradation is initiated by decapping and proceeds 5' to 3' behind the last translating ribosome. These results provide an explanation for a long standing mystery in the miRNA field.

A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation.

This protocol describes a method that uses splinted ligation for in-solution, direct labeling of small RNAs from total RNA. The liquid phase hybridization method makes it possible to achieve sensitive, specific, and quantitative detection while eliminating a number of time-consuming and labor-intensive steps required for the standard Northern blot assay. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5' end radiolabeled ligation oligonucleotide. The captured small RNA is internally labeled by ligation. Detection of the labeled small RNAs is performed by denaturing gel electrophoresis and autoradiography or phosphorimaging. This protocol has been successfully used to study expression of various classes of biological small RNAs from nanogram to microgram amounts of total RNA without an amplification step and is significantly more simple and more sensitive than Northern blotting or ribonuclease protection assays. Once the oligonucleotides have been synthesized and total RNA has been extracted, the procedure can be completed in 6 h.

Co-translational mRNA decay in Saccharomyces cerevisiae.

The rates of RNA decay and transcription determine the steady-state levels of all messenger RNA and both can be subject to regulation. Although the details of transcriptional regulation are becoming increasingly understood, the mechanism(s) controlling mRNA decay remain unclear. In yeast, a major pathway of mRNA decay begins with deadenylation followed by decapping and 5'-3' exonuclease digestion. Importantly, it is hypothesized that ribosomes must be removed from mRNA before transcripts are destroyed. Contrary to this prediction, here we show that decay takes place while mRNAs are associated with actively translating ribosomes. The data indicate that dissociation of ribosomes from mRNA is not a prerequisite for decay and we suggest that the 5'-3' polarity of mRNA degradation has evolved to ensure that the last translocating ribosome can complete translation.

Direct detection of small RNAs using splinted ligation.

This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5'-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ligation. Detection of the labeled small RNAs is performed by denaturing gel electrophoresis and autoradiography or phosphor-imaging. This protocol has been successfully used to study expression of various classes of biological small RNAs from nanogram to microgram amounts of total RNA without an amplification step. It is significantly simpler to perform and more sensitive than either northern blotting or ribonuclease protection assays. Once the oligonucleotides have been synthesized and total RNA has been extracted, the procedure can be completed in 6 h.

A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation.

The discovery and characterization of microRNAs (miRNAs) and other families of short RNAs has led to a rapid expansion of research directed at elucidating their expression patterns and regulatory functions. Here, we describe a convenient, sensitive, and straightforward method to detect and quantitate specific miRNA levels in unfractionated total RNA samples. The method, based on splinted ligation, does not require specialized equipment or any amplification step, and is significantly faster and more sensitive than Northern blotting. We demonstrate that the method can be used to detect various classes of small regulatory RNAs from different organisms.

Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways.

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves.

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

SH3 domain protein-binding arrays.

First identified as part of the Rous sarcoma oncogene product Src, SH3 (Src Homology 3) domains play an important role in intercellular communication and intracellular signal transduction. A high-throughput assay for ligand binding to SH3 domains--SH3 domain proteins immobilized on a membrane--allows rapid visualization of numerous SH3 domain protein-protein interactions with no expensive equipment or radioactivity required. Once the array is constructed or obtained commercially, the procedure is straightforward: The protein of interest is cloned into a fusion-tagged expression vector and expressed in bacteria, the prepared bacterial extract is incubated with the array membrane, and the signal is measured using a chemiluminescence detection system.

Mg2+ sensing by the Mg2+ sensor PhoQ of Salmonella enterica.

The PhoP/PhoQ two-component regulatory system governs the adaptation to low Mg(2+) environments and virulence in several Gram-negative species. During growth in low Mg(2+), the sensor PhoQ modifies the activity of the response regulator PhoP promoting gene transcription, whereas growth in high Mg(2+) represses transcription of PhoP-activated genes. The PhoQ protein harbors a periplasmic domain of 146 amino acid residues that binds Mg(2+) in vitro and is required for Mg(2+)-mediated repression in vivo. Here, we identify periplasmic mutants of the Salmonella PhoQ protein that allow transcription of PhoP-activated genes even under high Mg(2+) concentrations. When expressed in a strain harboring a PhoP variant that is phosphorylated from acetyl phosphate, some of the mutants failed to repress PhoP-promoted transcription in high Mg(2+), whereas others displayed a wild-type ability to do so. Mutant PhoQ proteins that allowed expression of PhoP-activated genes in high Mg(2+) displayed a pattern of iron-mediated cleavage in vitro that was different from that displayed by wild-type PhoQ, indicative of altered Mg(2+) binding. A PhoQ protein with the conserved histidine residue (H277) substituted by alanine could not promote transcription of PhoP-activated genes in low Mg(2+) but could turn off expression in response to high Mg(2+). Our studies demonstrate that residues G93, W97, H120 and T156 are required for a wild-type response to Mg(2+), and suggest that Mg(2+) binding to the periplasmic domain regulates several activities in the PhoQ protein.

Fe(III)-mediated cellular toxicity.

Because it can undergo reversible changes in oxidation state, iron is an excellent biocatalyst but also a potentially deleterious metal. Iron-mediated toxicity has been ascribed to Fe(II), which reacts with oxygen to generate free radicals that damage macromolecules and cause cell death. However, we now report that Fe(III) exhibits microbicidal activity towards strains of Salmonella enterica, Escherichia coli and Klebsiella pneumoniae defective in the Fe(III)-responding PmrA/PmrB signal transduction system. Fe(III) bound to a pmrA Salmonella mutant more effectively than to the isogenic wild-type strain and exerted its microbicidal activity even under anaerobic conditions. Moreover, Fe(III) permeabilized the outer membrane of the pmrA mutant, rendering it susceptible to vancomycin, which is normally non-toxic to Gram-negative species. On the other hand, Fe(III) did not affect the viability of a mutant defective in Fur, the major regulator of cytosolic iron homeostasis, which is hypersensitive to Fe(II)-mediated toxicity. A functional pmrA gene was necessary for bacterial survival in soil. Our results indicate that Fe(III) exerts its microbicidal activity by a mechanism that is oxygen independent and different from that mediated by Fe(II).

Mg2+ homeostasis and avoidance of metal toxicity.

Because Mg2+ is required for a wide variety of cellular functions, its intracellular levels must be tightly regulated. In the Gram-negative bacterium Salmonella enterica, three transporters mediate Mg2+ uptake: the P-type ATPases MgtA and MgtB, whose expression is transcriptionally induced in low Mg2+ by the Mg2+-regulated PhoP/PhoQ two-component system; and CorA, whose transcription is regulated neither by the levels of Mg2+ nor by the PhoP/PhoQ system. We now report that mutants defective in phoP or in both mgtA and mgtB are hypersensitive to oxidative stress-dependent Fe (II)-mediated killing. These mutants display increased iron accumulation and heightened Ni2+ uptake. Inactivation of the corA gene restored Fe(II) resistance to the phoP mutant and eliminated uptake of Ni2+. Neither corA transcription nor CorA protein levels were altered in the phoP mutant, suggesting that CorA alters its activity in response to the presence of PhoP-regulated determinants. Downregulation of CorA activity in low Mg2+ environments may enable Salmonella to avoid the uncontrolled influx of toxic metals.