Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression Here we investigate the requirements for structure and delivery of the interfering RNA To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference The effects of this interference were evident in both the injected animals and their progeny Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

https://www.auburn.edu/academic/classes/biol/6190/refs/L04/1-s2.0-S0092867402009716-main.pdf

Integrins: Bidirectional, Allosteric Signaling Machines

https://www.cell.com/cell/pdf/S0092-8674(00)80434-1.pdf

Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade

The SREBP Pathway: Regulation of Cholesterol Metabolism by Proteolysis of a Membrane-Bound Transcription Factor

https://www.univie.ac.at/ibmz/student/results/apoptosis1.pdf

Apaf-1, a Human Protein Homologous to C. elegans CED-4, Participates in Cytochrome c–Dependent Activation of Caspase-3

The structure of the G protein heterotrimer Giα1β1γ2

ACETYLCHOLINE released during parasv mpathetic stimulation of the vagal nerve slows the heart rate through the activation of muscarinic receptors and subsequent opening of an inwardly rectifying potassium channel1. The activation of these muscarinic potassium channels is mediated by a pertussis toxin-sensitive heterotrimeric GTP-binding protein (G protein)2,3. It has not been resolved whether exogenously applied Gα4,5 or Gβγ6,7, or both, activate the channel. Using a heterologous expression system, we have tested the ability of different G protein subunits to activate the cloned muscarinic potassium channel, GIRK18,9. We report here that coexpression of GIRK1 with Gβγ but not Gαβγ in Xenopus oocytes results in channel activity that persists in the absence of cytoplasmic GTP. This activity is reduced by fusion proteins of the (β-adrenergic receptor kinase and of recombinant Gαi–GDP, both of which are known to interact with Gβγ10,11. Moreover, application of recombinant Gβγ, but not Gαi–GTP-γS, activates GIRK1 channels. Thus Gβγ appears to be sufficient for the activation of GIRK1 muscarinic potassium channels.

Activation of the cloned muscarinic potassium channel by G protein βγ subunits

Acetylcholine activates inwardly rectifying potassium channels (IK.ACh) in the heart through muscarinic receptor binding and activation of pertussis-toxin-sensitive G proteins. Experiments showing that only the beta gamma-subunit (G beta gamma) activates IK.ACh (ref. 4) were challenged by reports that only the activated alpha-subunit (G alpha) was effective. Here we examine IK.ACh regulation using purified brain and recombinant G-protein subunits. Six recombinant G beta gamma-subunits activated IK.ACh with apparent half-maximal activation concentrations of 3-30 nM. Activation of IK.ACh by recombinant G alpha-GTP gamma S was observed, but this was probably due to release of GTP gamma S from the protein. Importantly, IK.ACh activity elicited by GTP gamma S was inhibited by purified brain and recombinant G alpha-GDP, suggesting that native G beta gamma plays a major role in this pathway. We conclude that G beta gamma is a primary regulator of IK.ACh activity.

Recombinant G-protein βγ-subunits activate the muscarinic-gated atrial potassium channel

Evidence suggests that both alpha and beta gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) inhibit adenylyl cyclase. Although type I adenylyl cyclase is inhibited directly by exogenous beta gamma, inhibition of adenylyl cyclase by Gi alpha has not been convincingly demonstrated in vitro. Concentration-dependent inhibition of adenylyl cyclases by purified Gi alpha subunits is described. Activated Gi alpha but not G(o) alpha was effective, and myristoylation of Gi alpha was required. The characteristics of the inhibitory effect were dependent on the type of adenylyl cyclase and the nature of the activator of the enzyme. The concentrations of Gi alpha required to inhibit adenylyl cyclase were substantially higher than those normally thought to be relevant physiologically. However, analysis indicates that these concentrations may be relevant and reasonable.

https://science.sciencemag.org/content/sci/261/5118/218.full.pdf

Jorge A. Iñiguez-Lluhi

Papers

The structure of the G protein heterotrimer Giα1β1γ2

Activation of the cloned muscarinic potassium channel by G protein βγ subunits

Recombinant G-protein βγ-subunits activate the muscarinic-gated atrial potassium channel

Inhibition of adenylyl cyclase by Gi alpha

G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma.