Final Report Summary - TIPTGSVSR (Biochemical characterization of RNA silencing mechanisms and their alteration by viral proteins in plant cell-free systems)
Summary for research objective 1: In vitro recapitulation and dissection of plant miRNA-mediated translational repression
(Objective 1.a) Recapitulation of AGO1- and AGO10-mediated translational repression (TR) using artificial and authentic sRNA:target interactions from Arabidopsis.
Several plant miRNAs including miR171, miR168, and miR834, have been characterized to mediate translational repression (TR) in vivo. To study plant TR in vitro, it was first tested if these miRNAs are efficiently loaded to AGO10 as well as AGO1 in BYL (evacuorated protoplast lysate of Nicotiana BY-2 cells). One of tested miRNAs, miR171, that targets SCL6 mRNA, was loaded into AGO1 and AGO10 in a comparative efficiency. On the other hand, unexpectedly, miR168 that is known to target AGO1 mRNA and induce TR in AGO10-dependent manner was not efficiently loaded into AGO10. The loading of miR834 that targets CIP4 mRNA was not efficient enough to detect AGO1- or AGO10-RISC assemblies. Notably, the rate-limiting step in miR834 loading was the star strand unwinding but not the binding of duplex. Different from miR834, the rate-limiting step in miR168 loading to AGO10 was the binding of duplex to AGO10. Based on these findings, miR171 was used for further experiments to study TR in BYL. When RISC was made with miR171, AGO1 and AGO10 in RISC showed similar slicer activity, i.e. the amount of miR171-loaded RISC correlates with the amount of cleaved target RNA. The result was consistent between artificial and authentic targets. These data do not support the idea that AGO10 exhibits TR activity more effectively than AGO1. To improve the accuracy of measurement of TR, in vitro translation and the repression by RISC were measured by 35S-methionine incorporation. However, clear signal corresponding to protein product from target mRNA was not obtained, possibly because the translation activity of BYL was attenuated during RISC loading. Further optimization of experimental conditions would enable accurate evaluation of the extent of TR mediated by RISC.
(Objective 1.b) Investigate the dependency of TR upon deadenylation, decapping.
This project did not reach the step to perform work on this objective. As mentioned above, this is because the development of TR system using BYL was not completed in the period. One of the barriers for the completion is that RISC assembly in BYL is not active enough to analyze TR clearly. In addition, immunoblot-based measurement of translation was not sensitive enough to evaluate the extent of TR. In order to solve this problem, the translation activity was measured by the incorporation of 35S-methionine.
(Objective 1.c) Isolate and characterize BYL-expressed AGO1/10 complexes engaged into pure TR versus combined TR-slicing using complexes of WT AGO1/AGO10 or mutant alleles either lacking catalysis or specifically defective in TR.
As mentioned above, AGO10 did not exhibit unique ability not to cleave target RNA and to induce TR, in comparison with AGO1. An AGO1 derivative expressed in ago1-27 allele has been characterized to mediate TR preferentially. The relevant AGO1 allele in Nicotiana was expressed in BYL. Unexpectedly, this allele showed significant impairment in RISC loading. Thus, this project did not identify AGO1/AGO10 complexes engaged into pure TR.
Summary for research objective 2: deciphering the molecular targets and modes of action of viral suppressors of RNA silencing (VSRs)
(Objective 2.a) Analyze comparatively the global effects of VSRs on dsRNA dicing, RISC assembly, and RISC-mediated slicing/TR.
To decipher the molecular targets and mode of actions of VSRs, Tomato bushy stunt virus (TBSV) P19, Potato virus Y (PVY) HC-Pro, Cucumber vein yellow virus (CVYV) P1b, Peanut clump virus (PCV) P15, Turnip crinkle virus (TCV) P38, and Cucumber mosaic virus (CMV) 2b were cloned and expressed in BYL by in vitro translation. RISC loading was inhibited by all the tested VSR, while not affected by the suppressor deficient alleles. This finding emphasizes that RISC loading is one of important steps targeted by VSRs to inactivate the antiviral RNA silencing activities. This project has developed an assay system to analyze dicing and the inhibition by VSR. In this system, radiolabeled dsRNA is processed by endogenous dicer proteins to generate 21 and 24-nt siRNA duplexes. It was found that TCV P38 uniquely exhibits dicing inhibition activity. Different from TCV P38, coat proteins of other members of carmovirus (Cardamine chlorotic fleck carmovirus and Pelargonium flower break carmovirus) did not exhibit RISC loading and dicing inhibition activities. This fact supports the idea that the suppressor functions of TCV P38 are independent of encapsidation of viral RNA as coat protein. To further provide deeper insight into the molecular mechanisms of TCV P38 to inhibit RISC loading and dicing, mutagenesis screening on P38 was performed. Critically, this screening identified 57th arginine (R) as a residue specifically required for the ability of P38 to inhibit RISC loading. Similar to 57th R, 74th R that has been genetically identified to be an essential residue for suppressor function of P38 in planta was found to be specifically required for RISC loading inhibition but not for dicing inhibition in BYL. Thus, point mutations on 57th and 74th R residues successfully discriminated the two abilities of P38, RISC loading inhibition and dicing inhibition. Affinity purification analyses demonstrated the physical interactions between P38 and siRNA duplex. The interaction was size-selective, i.e. P38 prefers 21-nt to 24-nt siRNAs. Mutations on 57th and 74th R residues impaired P38-siRNA duplex interaction, showing the strict correlation between RISC loading inhibition and P38-siRNA duplex interaction. In dicing inhibition, affinity purification analyses showed that P38 physically interacts with dsRNA substrate. Two residues (84th R and 122th E) of P38 were found to be required to interact with dsRNA and to inhibit dicing in BYL.
(Objective 2.b) Copurification of specific Dicers or AGO1&2&10 with VSR, and characterization of the isolated complexes and assay their significance by reverse genetics in Arabidopsis.
Considering the findings that TCVP38 uniquely inhibits dicing inhibition, mass spectrometry analyses have been performed to identify specific Dicers and the associated proteins in P38-mediated dicing inhibition. Preliminary results did not clearly list up the candidate proteins, and further optimization of experimental condition is required.
(Objective 2.c) Identify which precise AGO form(s) is targeted by VSRs.
As shown in planta, the physical interaction between TCV P38 and AGO1, and the impairment by mutations on GW motif of P38 to bind AGO1 were confirmed in BYL. Importantly, point mutations on 74th R and 122th E that abrogate the ability of P38 to inhibit RISC loading did not alter the affinity to AGO1, indicating that P38-AGO1 interaction alone is not sufficient for RISC loading inhibition. Rather, these results emphasize the importance of P38-sRNA duplex interaction. Considering this, this project did not focus more on P38-AGO1 physical interaction, but provided deeper insight into P38-sRNA duplex interaction.
Expected final results and the potential impact
This project has shown significant progress on the dissection of the action of TCV P38 as VSR. Further experiments based on mass spectrometry analysis could identify host proteins targeted by P38, especially in dicing inhibition. The inhibitor (VSR)-based dissection of plant RNA silencing pathways are not well documented, and the continuous study would give novel information that could not been gained by classical genetic approaches on RNA silencing.
(Objective 1.a) Recapitulation of AGO1- and AGO10-mediated translational repression (TR) using artificial and authentic sRNA:target interactions from Arabidopsis.
Several plant miRNAs including miR171, miR168, and miR834, have been characterized to mediate translational repression (TR) in vivo. To study plant TR in vitro, it was first tested if these miRNAs are efficiently loaded to AGO10 as well as AGO1 in BYL (evacuorated protoplast lysate of Nicotiana BY-2 cells). One of tested miRNAs, miR171, that targets SCL6 mRNA, was loaded into AGO1 and AGO10 in a comparative efficiency. On the other hand, unexpectedly, miR168 that is known to target AGO1 mRNA and induce TR in AGO10-dependent manner was not efficiently loaded into AGO10. The loading of miR834 that targets CIP4 mRNA was not efficient enough to detect AGO1- or AGO10-RISC assemblies. Notably, the rate-limiting step in miR834 loading was the star strand unwinding but not the binding of duplex. Different from miR834, the rate-limiting step in miR168 loading to AGO10 was the binding of duplex to AGO10. Based on these findings, miR171 was used for further experiments to study TR in BYL. When RISC was made with miR171, AGO1 and AGO10 in RISC showed similar slicer activity, i.e. the amount of miR171-loaded RISC correlates with the amount of cleaved target RNA. The result was consistent between artificial and authentic targets. These data do not support the idea that AGO10 exhibits TR activity more effectively than AGO1. To improve the accuracy of measurement of TR, in vitro translation and the repression by RISC were measured by 35S-methionine incorporation. However, clear signal corresponding to protein product from target mRNA was not obtained, possibly because the translation activity of BYL was attenuated during RISC loading. Further optimization of experimental conditions would enable accurate evaluation of the extent of TR mediated by RISC.
(Objective 1.b) Investigate the dependency of TR upon deadenylation, decapping.
This project did not reach the step to perform work on this objective. As mentioned above, this is because the development of TR system using BYL was not completed in the period. One of the barriers for the completion is that RISC assembly in BYL is not active enough to analyze TR clearly. In addition, immunoblot-based measurement of translation was not sensitive enough to evaluate the extent of TR. In order to solve this problem, the translation activity was measured by the incorporation of 35S-methionine.
(Objective 1.c) Isolate and characterize BYL-expressed AGO1/10 complexes engaged into pure TR versus combined TR-slicing using complexes of WT AGO1/AGO10 or mutant alleles either lacking catalysis or specifically defective in TR.
As mentioned above, AGO10 did not exhibit unique ability not to cleave target RNA and to induce TR, in comparison with AGO1. An AGO1 derivative expressed in ago1-27 allele has been characterized to mediate TR preferentially. The relevant AGO1 allele in Nicotiana was expressed in BYL. Unexpectedly, this allele showed significant impairment in RISC loading. Thus, this project did not identify AGO1/AGO10 complexes engaged into pure TR.
Summary for research objective 2: deciphering the molecular targets and modes of action of viral suppressors of RNA silencing (VSRs)
(Objective 2.a) Analyze comparatively the global effects of VSRs on dsRNA dicing, RISC assembly, and RISC-mediated slicing/TR.
To decipher the molecular targets and mode of actions of VSRs, Tomato bushy stunt virus (TBSV) P19, Potato virus Y (PVY) HC-Pro, Cucumber vein yellow virus (CVYV) P1b, Peanut clump virus (PCV) P15, Turnip crinkle virus (TCV) P38, and Cucumber mosaic virus (CMV) 2b were cloned and expressed in BYL by in vitro translation. RISC loading was inhibited by all the tested VSR, while not affected by the suppressor deficient alleles. This finding emphasizes that RISC loading is one of important steps targeted by VSRs to inactivate the antiviral RNA silencing activities. This project has developed an assay system to analyze dicing and the inhibition by VSR. In this system, radiolabeled dsRNA is processed by endogenous dicer proteins to generate 21 and 24-nt siRNA duplexes. It was found that TCV P38 uniquely exhibits dicing inhibition activity. Different from TCV P38, coat proteins of other members of carmovirus (Cardamine chlorotic fleck carmovirus and Pelargonium flower break carmovirus) did not exhibit RISC loading and dicing inhibition activities. This fact supports the idea that the suppressor functions of TCV P38 are independent of encapsidation of viral RNA as coat protein. To further provide deeper insight into the molecular mechanisms of TCV P38 to inhibit RISC loading and dicing, mutagenesis screening on P38 was performed. Critically, this screening identified 57th arginine (R) as a residue specifically required for the ability of P38 to inhibit RISC loading. Similar to 57th R, 74th R that has been genetically identified to be an essential residue for suppressor function of P38 in planta was found to be specifically required for RISC loading inhibition but not for dicing inhibition in BYL. Thus, point mutations on 57th and 74th R residues successfully discriminated the two abilities of P38, RISC loading inhibition and dicing inhibition. Affinity purification analyses demonstrated the physical interactions between P38 and siRNA duplex. The interaction was size-selective, i.e. P38 prefers 21-nt to 24-nt siRNAs. Mutations on 57th and 74th R residues impaired P38-siRNA duplex interaction, showing the strict correlation between RISC loading inhibition and P38-siRNA duplex interaction. In dicing inhibition, affinity purification analyses showed that P38 physically interacts with dsRNA substrate. Two residues (84th R and 122th E) of P38 were found to be required to interact with dsRNA and to inhibit dicing in BYL.
(Objective 2.b) Copurification of specific Dicers or AGO1&2&10 with VSR, and characterization of the isolated complexes and assay their significance by reverse genetics in Arabidopsis.
Considering the findings that TCVP38 uniquely inhibits dicing inhibition, mass spectrometry analyses have been performed to identify specific Dicers and the associated proteins in P38-mediated dicing inhibition. Preliminary results did not clearly list up the candidate proteins, and further optimization of experimental condition is required.
(Objective 2.c) Identify which precise AGO form(s) is targeted by VSRs.
As shown in planta, the physical interaction between TCV P38 and AGO1, and the impairment by mutations on GW motif of P38 to bind AGO1 were confirmed in BYL. Importantly, point mutations on 74th R and 122th E that abrogate the ability of P38 to inhibit RISC loading did not alter the affinity to AGO1, indicating that P38-AGO1 interaction alone is not sufficient for RISC loading inhibition. Rather, these results emphasize the importance of P38-sRNA duplex interaction. Considering this, this project did not focus more on P38-AGO1 physical interaction, but provided deeper insight into P38-sRNA duplex interaction.
Expected final results and the potential impact
This project has shown significant progress on the dissection of the action of TCV P38 as VSR. Further experiments based on mass spectrometry analysis could identify host proteins targeted by P38, especially in dicing inhibition. The inhibitor (VSR)-based dissection of plant RNA silencing pathways are not well documented, and the continuous study would give novel information that could not been gained by classical genetic approaches on RNA silencing.