Regulation of the activity of the promoter of RNA‐induced silencing, C3PO

Sahu, Shriya; Williams, Leo; Pérez, Alberto; Philip, Finly; Caso, Giuseppe; Zurawsky, Walter; Scarlata, Suzanne

doi:10.1002/pro.3219

Cited by 15 publications

(22 citation statements)

References 38 publications

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“…Down-regulating PLCβ1 resulted in a small peak at low molecular weights followed by a broad peak at larger sizes that shifts to the right when compared to control. The small peak is consistent with enhanced C3PO activity due relief of inhibition by PLCβ1 (25). .…”

Section: Cytosolic Plcβ1 Levels Impacts the Size Of Cytosolic Rnassupporting

confidence: 67%

“…In an initial study, we purified PLCβ1 and covalently labeled it with Alexa488 that served as a FRET donor. We then purified eIF5A, labeled it with a FRET acceptor, Alexa594, and measured the association between the two proteins in solution similar to previous studies (25). We find that the two proteins bind with strong affinity (Kd = 27 + 5nM).…”

Section: Plcβ1 Binds To Eif5a In the Same Region As C3po And Gαqmentioning

confidence: 55%

“…Sequence alignment of Ago2 and the TRAX subunits of C3PO shows four homologous regions ranging from ~20-40 aa in length and from 21-30% identity and 40-56% homology (2-54, 87-119, 202-228, 109-136 on C3PO and 788-826, 555-598, 188-202, 831-858 Ago2). It is notable that at least three of the TRAX regions are potential interaction sites for PLCβ1 binding and at least one of these may be available for PLCβ1-Ago2 binding (25). By this argument, it is possible that PLCβ1 directly binds Ago2 through similar interactions as TRAX.…”

Section: Discussionmentioning

confidence: 99%

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Stimulation of phospholipase Cβ1 by Gαq promotes the assembly of stress granule proteins

Qifti

Jackson

Singla

et al. 2019

Preprint

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During adverse environmental conditions, mammalian cells regulate protein production by sequestering the translation machinery in membraneless organelles (i.e. stress granules) whose formation is carefully regulated. In this study, we show a direct connection between G protein signaling and stress granule formation through phospholipase Cβ (PLCβ). In cells, PLCβ localizes to both the cytoplasm and plasma membrane. We find that a major population of cytosolic PLCβ binds to stress granule proteins; specifically, eIF5A and Ago2, whose RNA-induced silencing activity is halted under stress. PLCβ1 is activated by Gαq in response to hormones and neurotransmitters and we find that activation of Gαq shifts the cytosolic population of PLCβ1 to the plasma membrane, releasing stress granule proteins. This release is accompanied by the formation of intracellular particles containing stress granule markers, an increase in the size and number of particles, and a shift of cytosolic RNAs to larger sizes consistent with cessation of transcription. Arrest of protein synthesis is seen when the cytosolic level of PLCβ1 is lowered by siRNA or by osmotic stress, but not cold, heat or oxidative stress causes similar behavior. Our results fit a simple thermodynamic model in which eIF5a and its associated proteins partition into particles after release from PLCβ1 due to Gαq stimulation. Taken together, our studies show a link between Gαq-coupled signals and transcription through stress granule formation.

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Section: Cytosolic Plcβ1 Levels Impacts the Size Of Cytosolic Rnassupporting

confidence: 67%

Section: Plcβ1 Binds To Eif5a In the Same Region As C3po And Gαqmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

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Stimulation of phospholipase Cβ1 by Gαq promotes the assembly of stress granule proteins

Qifti

Jackson

Singla

et al. 2019

Preprint

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“…Part of the selectivity of the oligonucleotides that are silenced by PLCβ is dictated by the relative concentration of the specific miR, the presence of any competing miRs, and the stability of the miRs. Insight into the conditions that allow PLCβ to impact hydrolysis of specific oligonucleotides can be found in the thermodynamic cycle:…”

Section: Plcβ Inhibits the Promoter Of Rna‐induced Silencing C3po Tmentioning

confidence: 99%

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

et al. 2019

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Some proteins can serve multiple functions depending on different cellularconditions. An example of a bifunctional protein is inositide-specific mammalian phospholipase Cβ (PLCβ). PLCβ is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium. Recently, alternate cellular function(s) of PLCβ have become uncovered. However, the conditions that allow these different functions to be operative are unclear. Like many mammalian proteins, PLCβ has a conserved catalytic core along with several regulatory domains. These domains modulate the intensity and duration of calcium signals in response to external sensory information, and allow this enzyme to inhibit protein translation in a noncatalytic manner. In this review, we first describe PLCβ's cellular functions and regulation of the switching between these functions, and then discuss the thermodynamic considerations that offer insight into how cells manage multiple and competitive associations allowing them to rapidly shift between functional states.

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“…Support for this model comes from our live cell imaging data showing that hydrolysis of perfectly paired miR103-3 depends on the level of C3PO, PLCβ1 but imperfectly paired miR103-3 does not. It is notable that that PLCβ1 inhibits C3PO processing of miRs with higher Tms that are more slowly hydrolyzed by C3PO [15]. Thus, the impact of PLCβ1 on RNA-induced silencing by C3PO inhibition is limited to specific endogenous miRs.…”

Section: Discussionmentioning

confidence: 99%

Simulation of the Gαq /Phospholipase Cβ1 Signaling Pathway Returns Differentiated PC12 Cells to a Stem-like State

Garwain

Pearce

Jackson

et al. 2020

Preprint

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Phospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters, and is found at high levels in mammalian neuronal tissue. Besides carrying out this key plasma membrane function, PLCβ1 has a cytosolic population that helps, in part, to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCβ1 or reducing its cytosolic population by activating Gαq to drive it to the plasma membrane, returns differentiated PC12 cells to an undifferentiated state. In this state, the cells return to a spherical morphology, resume proliferation and express the stem cell transcription factors nanog and Oct4. Similar changes are seen with C3PO down-regulation. This return to a stem-like state is accompanied by shifts in multiple miR populations, such as increased levels of rno-miR-21 and rno-miR-26a. Surprisingly, we find that de-differentiation can also be induced by extended stimulation of the Gαq. In this case, the neurites completely retract over a 10-minute period, and while levels of nanog remain unchanged, the levels of some miRs begin to return to their undifferentiated values. In complementary studies, we followed the real time hydrolysis of a fluorescent-tagged miR in cells where PLCβ1 or C3PO were down-regulated. These samples showed substantial differences in miR processing in cells both the undifferentiated and differentiated states. Taken together, our studies suggest that PLCβ1, through its ability to regulate C3PO and endogenous miR populations, plays a key role in mediating PC12 cell differentiation.2 succession, some occur over longer periods resulting in cells that may have a narrow or broad a distribution of differentiation. Reversal of differentiation often occurs in cells that have transformed to cancerous states [1]. Thus, the ability to control differentiation and induce dedifferentiation would greatly aid in optimizing cancer therapies and other therapies where inducing or reversing differentiation would be beneficial.In this study, we show that we can manipulate the differentiated state of PC12 cells, a cultured cell line that is commonly used as a model for neuronal cells, through the Gαq/PLCβ1 signaling pathway [2,3]. This pathway is initiated when neurotransmitters such as acetylcholine, serotonin, dopamine and melatonin bind to their specific transmembrane receptor. The ligandbound receptor then activates Gαq which in turn activates phospholipase Cβ, leading to an increase in intracellular calcium. There are four known families of PLCβ, and PLCβ1 is the most responsive to Gαq, and is highly expressed in neuronal cells, such as PC12.Aside from its classic role on the plasma membrane, PLCβ1 also has an atypical cytosolic population that binds to unique partners. Specifically, we have found that PLCβ1 binds to and inhibits component 3 promoter of RISC activity (C3PO), which promotes RNA-induced silencing by accelerating degradation of the passenger strand of the silencing ...

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Regulation of the activity of the promoter of RNA‐induced silencing, C3PO

Cited by 15 publications

References 38 publications

Stimulation of phospholipase Cβ1 by Gαq promotes the assembly of stress granule proteins

Stimulation of phospholipase Cβ1 by Gαq promotes the assembly of stress granule proteins

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

Simulation of the Gαq /Phospholipase Cβ1 Signaling Pathway Returns Differentiated PC12 Cells to a Stem-like State

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