SnapShot: Mediator complex structure.

The Mediator complex controls RNA polymerase II transcription genome-wide. In humans, Mediator consists of 26 subunits; furthermore, a four-subunit "Mediator kinase module" can reversibly associate with the complex. Mediator structure is generally conserved from yeast to humans, although the human complex is larger, more structurally disordered, and contains metazoan-specific subunits. To view this SnapShot, open or download the PDF.

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

The Mediator complex as a master regulator of transcription by RNA polymerase II.

The Mediator complex, which in humans is 1.4 MDa in size and includes 26 subunits, controls many aspects of RNA polymerase II (Pol II) function. Apart from its size, a defining feature of Mediator is its intrinsic disorder and conformational flexibility, which contributes to its ability to undergo phase separation and to interact with a myriad of regulatory factors. In this Review, we discuss Mediator structure and function, with emphasis on recent cryogenic electron microscopy data of the 4.0-MDa transcription preinitiation complex. We further discuss how Mediator and sequence-specific DNA-binding transcription factors enable enhancer-dependent regulation of Pol II function at distal gene promoters, through the formation of molecular condensates (or transcription hubs) and chromatin loops. Mediator regulation of Pol II reinitiation is also discussed, in the context of transcription bursting. We propose a working model for Mediator function that combines experimental results and theoretical considerations related to enhancer-promoter interactions, which reconciles contradictory data regarding whether enhancer-promoter communication is direct or indirect. We conclude with a discussion of Mediator's potential as a therapeutic target and of future research directions.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Preparing scientists for a visual future: Visualization is a powerful tool for research and communication but requires training and support.

The increasing complexity of biological data along with the need to communicate results requires visualization. It requires more training and support though to help scientists create efficient visual representations of their work.

Lung injury pathways: Adenosine receptor 2B signaling limits development of ischemic bronchiolitis obliterans organizing pneumonia.

Purpose/Aim of the Study: Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. MATERIALS AND METHODS: Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague-Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strains containing a mutation in the A2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A2A (A2AAR) and A2B adenosine receptor (A2BAR) mRNA and protein were quantified. RESULTS: Twenty-four hours after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A2BAR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A2AAR mRNA and protein concentrations remained unchanged following ischemia. A2BAR protein was increased in PA ligated lungs of SS rats after 7 days, and 4 h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. CONCLUSION: Increased A2BAR and adenosine following unilateral lung ischemia as well as more BOOP in A2BAR mutant rats implicate a protective role for A2BAR signaling in countering ischemic lung injury.

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension.

The A(2B) adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A(2B)AR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A(2B)AR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15-20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A(2B)AR. Our data indicate varying roles for A(2B)AR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.

Monitoring seasonal variation of epicatechin and gallic acid in the bark of Saraca asoca using reverse phase high performance liquid chromatography (RP-HPLC) method.

BACKGROUND: Saraca asoca (Roxb.) Wilde (Fabaceae) is a high valued but vulnerable medicinal plant of Western Ghats region. This plant is mainly known for its use in various gynecological disorders. OBJECTIVE: The objective of the present study was to investigate seasonal variation of the polyphenolic compounds viz., epicatechin and gallic acid in the bark of S. asoca by using Reverse Phase High Performance Liquid Chromatography-Diode Array Detector (RP-HPLC-DAD) method. MATERIALS AND METHODS: The bark was collected in six different Ritu (season) viz. Varsha (monsoon), Sharad (autumn), hemant (early winter), Shishir (winter), Vasanta (spring), and Grishma (summer) mentioned in Ayurveda. RESULTS: The RP-HPLC-DAD analysis indicated that levels of epicatechin and gallic acid in the bark of S. asoca vary seasonally. The highest concentration of epicatechin was observed in Shishir Ritu (3315.19 ± 165.76 mg/100g) and gallic acid during Hemant Ritu (211.90 ± 10.60 mg/100 g). CONCLUSIONS: In present study, the ability to synthesize and accumulate both the compounds in bark of S. asoca varied greatly throughout the seasons. It was also observed that the compound epicatechin was present abundantly as compared to gallic acid throughout the seasons.

Root apoplastic barriers block Na+ transport to shoots in rice (Oryza sativa L.).

Rice is an important crop that is very sensitive to salinity. However, some varieties differ greatly in this feature, making investigations of salinity tolerance mechanisms possible. The cultivar Pokkali is salinity tolerant and is known to have more extensive hydrophobic barriers in its roots than does IR20, a more sensitive cultivar. These barriers located in the root endodermis and exodermis prevent the direct entry of external fluid into the stele. However, it is known that in the case of rice, these barriers are bypassed by most of the Na(+) that enters the shoot. Exposing plants to a moderate stress of 100 mM NaCl resulted in deposition of additional hydrophobic aliphatic suberin in both cultivars. The present study demonstrated that Pokkali roots have a lower permeability to water (measured using a pressure chamber) than those of IR20. Conditioning plants with 100 mM NaCl effectively reduced Na(+) accumulation in the shoot and improved survival of the plants when they were subsequently subjected to a lethal stress of 200 mM NaCl. The Na(+) accumulated during the conditioning period was rapidly released when the plants were returned to the control medium. It has been suggested that the location of the bypass flow is around young lateral roots, the early development of which disrupts the continuity of the endodermal and exodermal Casparian bands. However, in the present study, the observed increase in lateral root densities during stress in both cultivars did not correlate with bypass flow. Overall the data suggest that in rice roots Na(+) bypass flow is reduced by the deposition of apoplastic barriers, leading to improved plant survival under salt stress.