Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ

Sahu, Shriya; Philip, Finly; Scarlata, Suzanne

doi:10.1074/jbc.m113.531467

Cited by 19 publications

(33 citation statements)

References 29 publications

(43 reference statements)

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“…C3PO is an octamer consisting of 6 translin subunits that are responsible for the binding of specific oligonucleotides and 2 TRAX subunits that hydrolyze the bound oligonucleotides (7). We have found that PLCβ binds to an external site on one or both of the TRAX subunits in a 1:1 stoichiometric manner (13). Previously we observed that when we overexpressed TRAX in HEK293 cells that were overproducing both PLCβ1 and Gα q , we could quench the rise in intracellular Ca 2+ resulting from activation of Gα q ‐PLCβ (10).…”

Section: Resultsmentioning

confidence: 99%

RNA‐induced silencing attenuates G protein‐mediated calcium signals

et al. 2016

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Phospholipase Cβ (PLCβ) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCβ is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCβ raises the possibility that RNA silencing activity can affect the ability of PLCβ to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCβ stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCβ-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCβ-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals.

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Section: Resultsmentioning

confidence: 99%

RNA‐induced silencing attenuates G protein‐mediated calcium signals

et al. 2016

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“…We attached a fluorescent probe / quencher pair to the ends of different siRNA molecules to monitor hydrolysis of different sequences by C3PO in real time. Our studies found that C3PO hydrolyzes siRNA (GAPDH) at a much faster rate than siRNA (Hsp90) (Sahu, Philip, 2014). However, when PLCβ is bound to C3PO, the hydrolysis rate for siRNA (GAPDH) is significantly reduced while the rate for siRNA (Hsp90) is unchanged (see Fig.…”

Section: Topicsmentioning

confidence: 81%

“…However, when PLCβ is bound to C3PO, the hydrolysis rate for siRNA (GAPDH) is significantly reduced while the rate for siRNA (Hsp90) is unchanged (see Fig. 5, from (Sahu, Philip, 2014)).…”

Section: Topicsmentioning

confidence: 99%

“…One possible reason may be that cells contain different RISC complexes which have varying affinities to PLCβ, or that RISC processes RNAs differently depending on their structure / sequence which are differentially affected by PLCβ. We initially addressed this question by characterizing the biophysical properties of the C3PO-PLCβ complex (Sahu et al, 2014). C3PO crystallizes as an asymmetric octamer with two TRAX subunits and 6 translin subunits (Liu, Ye, 2009, Tian, Simanshu, 2011).…”

Section: Topicsmentioning

confidence: 99%

“…We have extensive molecular and cellular evidence that PLCβ directly binds to the TRAX subunits of C3PO and reduces the rate of hydrolysis of specific oligonucleotides, such as siRNA (GAPDH) (Sahu, Philip, 2014) to reverse RNA silencing (Philip, Guo, 2012) leading to changes in the genetic composition of the cell (Philip, Sahu, 2013) which may lead to changes in cell function such as differentiation. There is a possibility that other pathways induced by PLCβ play a role, although these potential pathways are not apparent at the moment.…”

Section: Topicsmentioning

confidence: 99%

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Phospholipase Cβ connects G protein signaling with RNA interference

Scarlata

Garwain

Williams

et al. 2016

Advances in Biological Regulation

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Phosphoinositide-specific-phospholipase Cβ (PLCβ) is the main effector of Gαq stimulation which is coupled to receptors that bind acetylcholine, bradykinin, dopamine, angiotensin II as well as other hormones and neurotransmitters. Using a yeast two-hybrid and other approaches, we have recently found that the same region of PLCβ that binds Gαq also interacts with Component 3 Promoter of RNA induced silencing complex (RISC) (C3PO), which is required for efficient activity of the RNA-induced silencing complex. In purified form, C3PO competes with Gαq for PLCβ binding and at high concentration can quench PLCβ activation. Additionally, we have found that the binding of PLCβ to C3PO inhibits its nuclease activity leading to reversal of RNA-induced silencing of specific genes. In cells, we found that PLCβ distributes between the plasma membrane where it localizes with Gαq, and in the cytosol where it localizes with C3PO. When cells are actively processing small interfering RNAs the interaction between PLCβ and C3PO gets stronger and leads to changes in the cellular distribution of PLCβ. The magnitude of attenuation is specific for different silencing RNAs. Our studies imply a direct link between calcium responses mediated through Gαq and post-transcriptional gene regulation through PLCβ.

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Kinesin‐1 inhibits the aggregation of amyloid‐β peptide as detected by fluorescence cross‐correlation spectroscopy

Zheng

Tian

et al. 2016

FEBS Letters

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Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-b (Ab) generated by the proteolytic processing of amyloid-b precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Ab generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Ab with kinesin-1 at the singlemolecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Ab over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

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Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ

Cited by 19 publications

References 29 publications

RNA‐induced silencing attenuates G protein‐mediated calcium signals

RNA‐induced silencing attenuates G protein‐mediated calcium signals

Phospholipase Cβ connects G protein signaling with RNA interference

Kinesin‐1 inhibits the aggregation of amyloid‐β peptide as detected by fluorescence cross‐correlation spectroscopy

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