Farnesylated heat shock protein 40 is a component of membrane-bound RISC in Arabidopsis

Abstract

ARGONAUTE1 (AGO1) binds directly to small regulatory RNA and is a key effector protein of post-transcriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA) in Arabidopsis. The formation of an RNA-induced silencing complex (RISC) of AGO1 and small RNA requires the function of the heat shock protein 70/90 chaperone system. Some functions of AGO1 occur in association with endomembranes, in particular the rough endoplasmic reticulum (RER), but proteins interacting with AGO1 in membrane fractions remain unidentified. In this study, we show that the farnesylated heat shock protein 40 homologs, J2 and J3, associate with AGO1 in membrane fractions in a manner that involves protein farnesylation. We also show that three changes in AGO1 function are detectable in mutants in protein farnesylation and J2/J3. First, perturbations of the HSP40/70/90 pathway by mutation of J3, HSP90, and farnesyl transferase affect the amounts of AGO1 associated with membranes. Second, miRNA association with membrane-bound polysomes is increased in farnesyl transferase and farnesylation-deficient J2/J3 mutants. Third, silencing by noncell autonomously acting short interfering RNAs is impaired. These observations highlight the involvement of farnesylated J2/J3 in small RNA-mediated gene regulation, and suggest that the importance of chaperone-AGO1 interaction is not limited to the RISC assembly process.

Keep calm and carry on: miRNA biogenesis under stress

“…In addition, some of the few proteins known to interact with AGO1 in plants are also heavily regulated by stress. HSP40/70/90, essential chaperons for the RISC assembly and AGO1 membrane association, are regulated under stressful conditions, potentially limiting the activity of the pathway under certain conditions (Barghetti et al ., ; Sjogren et al ., ). In the same way, the stress‐responsive ubiquitin E3 ligase HOS1 controls AGO1 levels by regulating miR168 transcription, which in turn is able to target and silence AGO1 mRNA (Wang et al ., ).…”

Genome-wide analysis of the rice J-protein family: identification, genomic organization, and expression profiles under multiple stresses

“…Furthermore, J-proteins not only function as co-chaperones in various biological processes (Miernyk 2001), but also act as enzymes or epigenetic regulators (De et al 1995; Richly et al 2010). In A. thaliana , the farnesylated J2 and J3 associate with AGO1 in membrane fractions in a manner that involves protein farnesylation, and also influences the distribution of miRNA between polysome-bound and unbound fraction (Sjögren et al 2018); the J-proteins embryo sac development arrest 3 (EDA3) and thermosensitive male sterile 1 ( TMS1 ) are implicated in the thermotolerance of pollen tubes (Valencia-Morales et al 2012; Yang et al 2009); the flowering time is regulated by AtJ3 via its direct binding to a MADS-box transcription factor (Shen et al 2011), and AtJ8 , AtJ11, and AtJ20 are involved in the optimization of photosynthetic reactions and stabilization of photosystem II (PSII) complexes under high light stress (Chen et al 2010). In tomato, LeCDJ1 is also found to be essential for maintaining PSII under chilling stress in tomato (Kong et al 2014b), and its J-domain is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance (Barghetti et al 2017).…”

Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

“…This is also in agreement with our previous results indicating that a prolonged AGO1–target interaction with a non-cleavable mRNA would favor AGO1 and miRNA degradation [ 33 ], which could in turn inhibit or hinder further processing of the loaded miRNA. The AGO 1 membrane association is, at least partially, dependent on farnesylation of the Heat Shock Protein 40 chaperones J3 and J2, two AGO1 interactors [ 51 ]. AGO1 levels in microsomes is reduced not only in j3/j2 mutants but also in mutants of the farnesyl transferase ERA1.…”