TY - BOOK
T1 - Investigating membrane-bound Argonaute functions in Arabidopsis
AU - Barghetti, Andrea
PY - 2017
Y1 - 2017
N2 - Small non-coding RNAs (sRNA) are key transcriptional and post-transcriptional regulators of geneexpression in plants and animals. In plants, they most commonly derive from double-stranded RNA(dsRNA) substrate processed by several ribonucleases type III DICER-LIKE (DCLs) enzymes into 20to 24 nucleotides long RNA duplexes. A major important class of sRNA is constituted by microRNA(miRNA), short endogenous hairpin-forming transcripts processed by DCL1. Small RNA and miRNAsilencing pathways play essential roles in regulating plant development and response to both biotic andabiotic stresses.The key effectors of sRNA-guided gene regulation are ARGONAUTE (AGO) proteins. A group ofHeat Shock Proteins of the HSP70/HSP90 chaperone machinery mediates the process, termed loading,that allow the functional association of sRNA with AGOs. Upon loading, Argonautes regulatecomplementary mRNA targets by direct cleavage or by inhibition of their translation.Arabidopsis genome encode for 10 different AGO proteins, of which AGO1 is the main effector ofpost-transcriptional gene silencing mediated by sRNA. Importantly, a fraction of AGO1 population isassociated with endomembranes, in particular with the rough endoplasmic reticulum (rER). Membranelocalizedargonaute functions include translational repression, production of secondary phased smallinterfering RNA (siRNA) and autophagy-mediated turnover. However proteins interacting with AGO1specifically on membrane fractions have not been identified and how AGO1 membrane recruitment ismediated as well as its functional importance remain poorly characterized.Isoprenoid biogenesis was previously found to be required for both AGO1 activity and membraneassociation, but the mechanistic connection between the two pathways was not discovered. Sinceprotein prenyl-transferases post-transcriptionally modify target proteins by attaching a hydrophobicprenyl group that can anchor them to membranes, they were suitable candidate regulators of AGO1membrane association.In this study we show that protein farnesyl transferase ERA1 can affect both activity and accumulationof membrane-bound AGO1 through farnesylation of the Heat Shock Protein 40 homologs J2 and J3,which we find to associate with AGO1 specifically on membranes. We also show that defectivefarnesylation of J2/J3 is the cause of several important developmental phenotypes observed in farnesyl transferase mutants, including ABA hypersensitivity, drought resistance, late flowering, enlargedmeristems and a newly discovered mis-regulation of a set of abiotic stress-related miRNAs.Finally we isolated and identified a new mutant in which membrane, but not soluble, compartments aredepleted of AGO1, and we utilize this mutant to investigate the consequences that such defect mayhave on AGO1 functions.
AB - Small non-coding RNAs (sRNA) are key transcriptional and post-transcriptional regulators of geneexpression in plants and animals. In plants, they most commonly derive from double-stranded RNA(dsRNA) substrate processed by several ribonucleases type III DICER-LIKE (DCLs) enzymes into 20to 24 nucleotides long RNA duplexes. A major important class of sRNA is constituted by microRNA(miRNA), short endogenous hairpin-forming transcripts processed by DCL1. Small RNA and miRNAsilencing pathways play essential roles in regulating plant development and response to both biotic andabiotic stresses.The key effectors of sRNA-guided gene regulation are ARGONAUTE (AGO) proteins. A group ofHeat Shock Proteins of the HSP70/HSP90 chaperone machinery mediates the process, termed loading,that allow the functional association of sRNA with AGOs. Upon loading, Argonautes regulatecomplementary mRNA targets by direct cleavage or by inhibition of their translation.Arabidopsis genome encode for 10 different AGO proteins, of which AGO1 is the main effector ofpost-transcriptional gene silencing mediated by sRNA. Importantly, a fraction of AGO1 population isassociated with endomembranes, in particular with the rough endoplasmic reticulum (rER). Membranelocalizedargonaute functions include translational repression, production of secondary phased smallinterfering RNA (siRNA) and autophagy-mediated turnover. However proteins interacting with AGO1specifically on membrane fractions have not been identified and how AGO1 membrane recruitment ismediated as well as its functional importance remain poorly characterized.Isoprenoid biogenesis was previously found to be required for both AGO1 activity and membraneassociation, but the mechanistic connection between the two pathways was not discovered. Sinceprotein prenyl-transferases post-transcriptionally modify target proteins by attaching a hydrophobicprenyl group that can anchor them to membranes, they were suitable candidate regulators of AGO1membrane association.In this study we show that protein farnesyl transferase ERA1 can affect both activity and accumulationof membrane-bound AGO1 through farnesylation of the Heat Shock Protein 40 homologs J2 and J3,which we find to associate with AGO1 specifically on membranes. We also show that defectivefarnesylation of J2/J3 is the cause of several important developmental phenotypes observed in farnesyl transferase mutants, including ABA hypersensitivity, drought resistance, late flowering, enlargedmeristems and a newly discovered mis-regulation of a set of abiotic stress-related miRNAs.Finally we isolated and identified a new mutant in which membrane, but not soluble, compartments aredepleted of AGO1, and we utilize this mutant to investigate the consequences that such defect mayhave on AGO1 functions.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01010614811&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - Investigating membrane-bound Argonaute functions in Arabidopsis
PB - Department of Biology, Faculty of Science, University of Copenhagen
ER -