Final Report Summary - PASRNA (Deciphering post-translational control of Argonautes and their effects on small RNA homeostasis (PASRNA))
Deciphering post-translational control of Argonautes and their effects on small RNA homeostasis
In plants and metazoans, non-coding RNAs, 19-24 nucleotides in length, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), have emerged as crucial transcriptional and post-transcriptional gene regulators, affecting processes as fundamental as genome integrity or cellular identity. miRNAs and siRNAs guide Argonaute (AGO) effector proteins to silence complementary RNA or DNA via various mechanisms. miRNAs, in particular, influence many biological processes including biotic/nonbiotic stress, immune responses, hormonal regulation, phase transitioning and patterning. miRNAs alone might control up to 30% of the transcriptional output of mammalian cells, and their unique expression signatures can be readily used to discriminate cell types. Stringent regulation of the small RNA pathways is thus a cellular necessity, as perturbations to expression or activity of these molecules may have considerably deleterious effects that include genetic instability, sterility and loss of cellular identity; genetic lesions in small RNA genes have also been implicated in several human disorders, to the point that many miRNAs are currently used as markers for cancer and other diseases. Thus, precise homeostatic control of miRNAs could be fundamental in guarding cells against malignancy. Shortly after their discovery, cellular repertoires of miRNAs were established and novel interactors of AGO isolated to establish the core of the RNA-induced silencing complex (RISC). However, contemporary small RNA biology has entered a more integrative, quantitative phase, adapted to the inherent complexity and interconnectivity of small RNA networks. In particular, identifying the regulatory mechanisms that underpin small RNA homeostasis by modulation of their biogenesis and action has become a key issue to be addressed, not only at the cellular but also organismal levels. Fortunately, small RNAs from plants (whole organisms) and cultured mammalian cells share commonalities in their biogenesis and action, thus we took advantage of a transversal and integrative approach using both model systems to investigate RNA silencing homeostasis, the major objective of this proposal.
An emerging theme in the homeostatic control of mammalian AGO stability and activity is through posttranslational modifications (PTM) affecting amino acid side-chains in a site-specific manner. PTM can temporarily or permanently enhance or disable the functionality and/or stability of target proteins by recruiting auxiliary factors, modify their subcellular localization, or induce their degradation. The following sections will summarise our insights into two novel Argonaute PTMs namely phosphorylation and ubiquitination and go on to highlight the intriguing roles for each which we have defined.
Employing a Biochemical screen, we have successfully identified novel phosphorylation events upon Argonaute which have the potential to be conserved in both plants and human. We have identified that these modifications, acting in response to external stimuli, facilitate rapid reprogramming of the loaded cellular microRNA content of the cell, which directly impacts upon the differentiation fate and thus function of a cell. We initially mapped these PTMs, with mass spectrometry, to the PIWI domain, the point of interface between AGO, loaded miRNA and targeted mRNA, furthermore, this domain provides the catalytic core of AGO. Our crystal structure analysis revealed that upon phosphorylation, these residues would be in direct contact with the phosphate backbone of the bound miRNA, creating a positively charged environment, prohibiting the binding of small RNAs and thus disabling AGOs ability to perform translational silencing. We hypothesised that phosphorylation may act as a rapid switch, to transiently unload Argonaute and provided a potentially short, but necessary, interval to re-establish a new cellular repertoire of loaded miRNAs, which could facilitate control of genome reprogramming. This could occur in response to a stress, internal/external stimuli and/or differentiation signals. We thus sought to identify a biological setting that initiates AGO phosphorylation. To fulfil this goal we successfully raised an antibody that could specifically recognise the phosphorylated form of Argonaute and with this we set out to determine the physiological conditions. Macrophages presented an ideal option to investigate the mechanism and consequences of rapid reprogramming of the miRNA portfolio. Macrophages play a highly versatile role in both the innate and adaptive immune response. In the presence of pathogenic stimuli, such as bacteria, naïve macrophages undergo a rapid transformation to become effective phagocytes; In support of this rapid and drastic expression changes of many microRNAs have been observed. Simultaneously, macrophages emit cytokines, which are necessary for the proper development of the inflammation response. Many cytokines so far have been shown, to be under the control of one or more miRNAs. Taken together this exemplifies a requirement for re-establishing the AGO bound miRNA portfolio upon macrophage activation and potentially in an AGO phosphorylation dependent manner. We discovered by treating both primary or transformed macrophages cell lines with lipopolysaccharide (LPS) a polysaccharide used to mimic bacterial infection through Toll Like receptors (TLR) activation that AGO was phosphorylated. We observed within 1 hour the occurrence of phosphorylation, this depleted in a time dependent manner over an 8-hour period. Similarly, within 1 hour, AGOs ability to bind small RNAs was severely reduced, this to was alleviated in a time dependent manner, further supporting that AGO phosphorylation status, is a key determinant in AGOs ability to bind small RNAs. Utilizing dual luciferase assays to determine AGOs ability to perform translation repression, in the presence of LPS, and other TLR ligands, we confirmed relieved translational repression upon macrophage activation, at early periods (<8hours). However, this was observed as a transient phenomenon as reversion to normal repression levels were observed after prolonged LPS exposure (>8hours). We are now currently investigating the impact of AGO phosphorylation on the inflammatory response our preliminary results suggest that miRNAs play a crucial role not only in the initial triggering of pro inflammation but in a subsequent orchestrated response to develop endotoxin tolerance to prohibit the cellular damage associated with continuous inflammation.
Previous studies in both animal and plant model systems have demonstrated an inherent link between AGO levels and the steady state expression of global populations of miRNAs, strongly suggesting a layer of homeostatic control Investigations during the project identified that AGOs is a substrate for ubiquitination and targeted for proteasome-mediated degradation. CUL RING E3 Ubiquitin Ligases (CRL) represents the largest class of human E3 ligases; they regulate an array of cellular processes from cell cycle progression and development to immunity via a large repertoire of substrates, including, potentially, AGOs. In an effort to uncover and characterise the mechanisms underlying AGO post-translational control, we employed MLN4924, a novel chemical inhibitor of CRL activation that blocks ubiquitination of known substrates. In the presence of MLN4924 we observed a marked increase in human AGO protein, but not mRNA, levels in a concentration-and time-dependent manner, suggesting that CRL(s) directly interact with AGO. Moreover AGOs ubiquitination profile was significantly reduced. We ascertained that prior stabilisation/accumulation of AGO results in an increase of endogenous miRNA steady state levels, confounding the hypothesis that AGO impacts on miRNA homeostasis. Luciferase reporter assays based on the activity of exogenous siRNAs revealed that prior CRL inhibition significantly enhanced RNAi. We have recent data to suggest that upon polarization of naïve T cells in to T helper cells AGO presence dictates the polarization route and in certain cases it would be desirable to increase AGO turnover. We are now in the process of creating a Retroviral transfection scheme to express either wildtype AGO ubiquitin mutant to test out this hypothesis and determine which routes require lose of AGO.
Collectively our results illuminate two novel mechanisms, whereby PTMs activity underpins AGO function, stability and small RNA homeostasis. This provides greater insight into fundamental aspects of small RNA biology; this is pivotal to further our knowledge regarding the therapeutic, veterinary and agronomical exploitation of these molecules.
In plants and metazoans, non-coding RNAs, 19-24 nucleotides in length, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), have emerged as crucial transcriptional and post-transcriptional gene regulators, affecting processes as fundamental as genome integrity or cellular identity. miRNAs and siRNAs guide Argonaute (AGO) effector proteins to silence complementary RNA or DNA via various mechanisms. miRNAs, in particular, influence many biological processes including biotic/nonbiotic stress, immune responses, hormonal regulation, phase transitioning and patterning. miRNAs alone might control up to 30% of the transcriptional output of mammalian cells, and their unique expression signatures can be readily used to discriminate cell types. Stringent regulation of the small RNA pathways is thus a cellular necessity, as perturbations to expression or activity of these molecules may have considerably deleterious effects that include genetic instability, sterility and loss of cellular identity; genetic lesions in small RNA genes have also been implicated in several human disorders, to the point that many miRNAs are currently used as markers for cancer and other diseases. Thus, precise homeostatic control of miRNAs could be fundamental in guarding cells against malignancy. Shortly after their discovery, cellular repertoires of miRNAs were established and novel interactors of AGO isolated to establish the core of the RNA-induced silencing complex (RISC). However, contemporary small RNA biology has entered a more integrative, quantitative phase, adapted to the inherent complexity and interconnectivity of small RNA networks. In particular, identifying the regulatory mechanisms that underpin small RNA homeostasis by modulation of their biogenesis and action has become a key issue to be addressed, not only at the cellular but also organismal levels. Fortunately, small RNAs from plants (whole organisms) and cultured mammalian cells share commonalities in their biogenesis and action, thus we took advantage of a transversal and integrative approach using both model systems to investigate RNA silencing homeostasis, the major objective of this proposal.
An emerging theme in the homeostatic control of mammalian AGO stability and activity is through posttranslational modifications (PTM) affecting amino acid side-chains in a site-specific manner. PTM can temporarily or permanently enhance or disable the functionality and/or stability of target proteins by recruiting auxiliary factors, modify their subcellular localization, or induce their degradation. The following sections will summarise our insights into two novel Argonaute PTMs namely phosphorylation and ubiquitination and go on to highlight the intriguing roles for each which we have defined.
Employing a Biochemical screen, we have successfully identified novel phosphorylation events upon Argonaute which have the potential to be conserved in both plants and human. We have identified that these modifications, acting in response to external stimuli, facilitate rapid reprogramming of the loaded cellular microRNA content of the cell, which directly impacts upon the differentiation fate and thus function of a cell. We initially mapped these PTMs, with mass spectrometry, to the PIWI domain, the point of interface between AGO, loaded miRNA and targeted mRNA, furthermore, this domain provides the catalytic core of AGO. Our crystal structure analysis revealed that upon phosphorylation, these residues would be in direct contact with the phosphate backbone of the bound miRNA, creating a positively charged environment, prohibiting the binding of small RNAs and thus disabling AGOs ability to perform translational silencing. We hypothesised that phosphorylation may act as a rapid switch, to transiently unload Argonaute and provided a potentially short, but necessary, interval to re-establish a new cellular repertoire of loaded miRNAs, which could facilitate control of genome reprogramming. This could occur in response to a stress, internal/external stimuli and/or differentiation signals. We thus sought to identify a biological setting that initiates AGO phosphorylation. To fulfil this goal we successfully raised an antibody that could specifically recognise the phosphorylated form of Argonaute and with this we set out to determine the physiological conditions. Macrophages presented an ideal option to investigate the mechanism and consequences of rapid reprogramming of the miRNA portfolio. Macrophages play a highly versatile role in both the innate and adaptive immune response. In the presence of pathogenic stimuli, such as bacteria, naïve macrophages undergo a rapid transformation to become effective phagocytes; In support of this rapid and drastic expression changes of many microRNAs have been observed. Simultaneously, macrophages emit cytokines, which are necessary for the proper development of the inflammation response. Many cytokines so far have been shown, to be under the control of one or more miRNAs. Taken together this exemplifies a requirement for re-establishing the AGO bound miRNA portfolio upon macrophage activation and potentially in an AGO phosphorylation dependent manner. We discovered by treating both primary or transformed macrophages cell lines with lipopolysaccharide (LPS) a polysaccharide used to mimic bacterial infection through Toll Like receptors (TLR) activation that AGO was phosphorylated. We observed within 1 hour the occurrence of phosphorylation, this depleted in a time dependent manner over an 8-hour period. Similarly, within 1 hour, AGOs ability to bind small RNAs was severely reduced, this to was alleviated in a time dependent manner, further supporting that AGO phosphorylation status, is a key determinant in AGOs ability to bind small RNAs. Utilizing dual luciferase assays to determine AGOs ability to perform translation repression, in the presence of LPS, and other TLR ligands, we confirmed relieved translational repression upon macrophage activation, at early periods (<8hours). However, this was observed as a transient phenomenon as reversion to normal repression levels were observed after prolonged LPS exposure (>8hours). We are now currently investigating the impact of AGO phosphorylation on the inflammatory response our preliminary results suggest that miRNAs play a crucial role not only in the initial triggering of pro inflammation but in a subsequent orchestrated response to develop endotoxin tolerance to prohibit the cellular damage associated with continuous inflammation.
Previous studies in both animal and plant model systems have demonstrated an inherent link between AGO levels and the steady state expression of global populations of miRNAs, strongly suggesting a layer of homeostatic control Investigations during the project identified that AGOs is a substrate for ubiquitination and targeted for proteasome-mediated degradation. CUL RING E3 Ubiquitin Ligases (CRL) represents the largest class of human E3 ligases; they regulate an array of cellular processes from cell cycle progression and development to immunity via a large repertoire of substrates, including, potentially, AGOs. In an effort to uncover and characterise the mechanisms underlying AGO post-translational control, we employed MLN4924, a novel chemical inhibitor of CRL activation that blocks ubiquitination of known substrates. In the presence of MLN4924 we observed a marked increase in human AGO protein, but not mRNA, levels in a concentration-and time-dependent manner, suggesting that CRL(s) directly interact with AGO. Moreover AGOs ubiquitination profile was significantly reduced. We ascertained that prior stabilisation/accumulation of AGO results in an increase of endogenous miRNA steady state levels, confounding the hypothesis that AGO impacts on miRNA homeostasis. Luciferase reporter assays based on the activity of exogenous siRNAs revealed that prior CRL inhibition significantly enhanced RNAi. We have recent data to suggest that upon polarization of naïve T cells in to T helper cells AGO presence dictates the polarization route and in certain cases it would be desirable to increase AGO turnover. We are now in the process of creating a Retroviral transfection scheme to express either wildtype AGO ubiquitin mutant to test out this hypothesis and determine which routes require lose of AGO.
Collectively our results illuminate two novel mechanisms, whereby PTMs activity underpins AGO function, stability and small RNA homeostasis. This provides greater insight into fundamental aspects of small RNA biology; this is pivotal to further our knowledge regarding the therapeutic, veterinary and agronomical exploitation of these molecules.