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.

Keywords: ARGONAUTE; Arabidopsis thaliana; Argonaute; Chaperones; Protein farnesylation; Rough Endoplasmic Reticulum; Small RNA; chaperone DnaJ (DnaJ); membrane; microRNA mechanism.

Figure 1.

Farnesyl transferase mutants show defects related to small RNA pathways. A, cotyledon, leaf, and inflorescence phenotypes of Col-0 WT, era1–2, dcl1–11, and era1–2/dcl1–11 mutants. B, relative mRNA expression levels of three miRNA targets encoding transcription factors (PHB, REV, and MYB65), as well as direct targets of these transcription factors. The figure shows results from a single biological replicate in which RNA from each genotype was prepared from pools of 12 adult leaves. Error bars indicate standard error in technical triplicates. Similar results were obtained when the entire experiment was repeated at another point in time. C, left, WT and era1–2 plants expressing the SUC:SUL (SS) hairpin. Right, ChlI (SUL) siRNA and protein levels of the individual plants shown on the left; total RNA fractions were analyzed by small RNA Northern blotting with a ChlI (SUL) probe. Total protein fractions were analyzed by Western blotting developed with ChlI antibodies. D, bottom, WT, era1–2 and plp-3 plants expressing the SUC:SUL (SS) hairpin. Top, ChlI (SUL) siRNA levels in leaves from pools of 5 plants from the F3 generation; total RNA fractions were analyzed by small RNA Northern blotting with a ChlI (SUL) probe.

Figure 2.

AGO1 interacts with farnesylated J2/J3 in membrane fractions. A, list of CAAX motif proteins identified in AGO1, but not in mock immunopurifications from deoxycholate-solubilized microsome fractions prepared from formaldehyde cross-linked 16-day-old seedling tissue. AGO1 is included to document efficiency of the purification. A list of all proteins identified is provided under the supporting Information. B, co-immunoprecipitation analysis of AGO1 and J2/J3 from membrane fractions. HCHO indicates formaldehyde cross-linking of seedling tissue prior to lysis. Mock, nonfunctional rabbit IgG applied in the same concentration as AGO1 antibody. C, co-immunoprecipitation analysis of AGO1 and HSP40 from membrane and soluble fractions. Noncross-linked tissue from 16-day-old seedlings was used. The samples were loaded on the same gel; white separation lines indicate the presence of additional lanes on the original gel. D, Western blot analysis of FLAG immunoprecipitates prepared from deoxycholate-solubilized membrane fractions obtained from an FHA-J3 transgenic line. Noncross-linked tissue from 16-day-old seedlings was used. Mock, parallel FLAG immunoprecipitation from nontransgenic parental line (Col-0). The samples were loaded on the same gel; white separation lines indicate the presence of additional lanes on the original gel.

Figure 3.

Farnesylation of J2/J3 is required for AGO1 interaction. A–C, AGO1 immunoprecipitates from deoxycholate-solubilized microsome fractions (16-day-old seedlings) analyzed by AGO1 and J2/J3 antibodies. Inputs were adjusted to ensure equal recovery of AGO1 in the immunoprecipitations.

Figure 4.

Mutation of J3 and HSP90 chaperones affects membrane association of AGO1. A and B, Western blotting of AGO1, J2/J3, HSP70, and HSP90 in total and microsome fractions prepared from inflorescence lysates of the indicated genotypes. For total fractions, equal loading was verified by Coomassie staining (CBB); for microsome fractions, Western blots were probed with SIP2 antibodies. Different sections of the membranes used for analysis of total lysates and microsome fractions were used for verification of protein loading; the leftmost molecular weight standard refers to the membrane used for total lysates, whereas the rightmost molecular weight standard refers to the membrane used for microsome fractions.

Figure 5.

Farnesylation of J3 affects membrane association of AGO1. A–D, Western blotting of AGO1, J2/J3, HSP70, and HSP90 in total and microsome fractions prepared from inflorescence lysates of the indicated genotypes. For total fractions, equal loading was verified by Coomassie staining (CBB); for microsome fractions, Western blots were probed with SIP2 antibodies. In A–C, different sections of the membranes used for analysis of total lysates and microsome fractions were used for verification of protein loading. In these cases, the leftmost molecular weight standard refers to the membrane used for total lysates, whereas the rightmost molecular weight standard refers to the membrane used for microsome fractions. E, RNase sensitivity of membrane-bound AGO1. Western blotting showing AGO1 abundance in microsome fractions with or without treatment with RNase A (10 μg/ml). Top, RNase A was added to hypertonic lysis buffer and was present throughout microsomal fractionation. Bottom, microsomes were prepared with RNase-free buffer, but were resuspended and washed for 15 min in lysis buffer with or without 10 μg/ml of RNase A.

Figure 6.

Farnesylation of J2/J3 does not affect AGO1 localization. A, analysis of microsome fractions obtained from 12-day-old liquid-culture grown seedlings by 20–50% sucrose gradient centrifugation. Aliquots of sucrose gradient fractions were analyzed by Western blotting with antibodies against the indicated proteins. MgCl₂, microsomes resuspended in buffer containing 5 mm MgCl₂; EDTA, microsomes resuspended in buffer containing 2 mm EDTA. B, AGO1 membrane IPs were prepared in the absence of detergent. Immunoprecipitates were analyzed by Western blots with the indicated antibodies. Microsomes resuspended in buffer without detergent were used as inputs.

Figure 7.

Membrane-bound RISC is loaded in the absence of J2/J3 farnesylation. A, AGO1 immunoprecipitates from soluble and membrane fractions analyzed by small RNA Northern blots. An aliquot of the immunoprecipitate was used for AGO1 Western blots to document equal recovery of the AGO1 protein from the different samples. The same Northern membrane was used for consecutive hybridizations to all probes. B, small RNA-seq analysis of small RNA isolated from AGO1 immunoprecipitates from microsome fractions. Ratios of read counts of pairs of miRNA/miRNA* are depicted for the indicated genotypes.

Figure 8.

Increased miRNA association with membrane-bound polysomes in era1 and j2/j3+J3^C417S. A and B, top, absorbance measured at 260 nm as a function of fraction number from a sucrose gradient used to separate polysome- and monosome-containing fractions from lighter fractions of microsome pellets isolated from the indicated mutant or transgenic lines. Bottom, small RNA Northern blot analysis of miRNA levels in total RNA (TOT) and RNA extracted from total microsomal (micro), monosome-containing (mono) and polysome-containing (poly) microsomal fractions. Several polysome-containing fractions were pooled as indicated on the top left panel in A.

Figure 9.

Tentative model summarizing the results on J2/J3 farnesylation and membrane-bound AGO1. The model puts forward the idea that the HSP40/70/90 pathway, and in particular farnesylated J2/J3, may engage in multiple functional interactions with membrane-bound AGO1. These may include promotion of membrane association of J3 (40) and AGO1 interaction (this work), AGO1 loading (6), the amount of polysome-bound RISC (this work), and, perhaps, a role in regulated proteolysis (not directly supported by evidence, but consistent with observations reported here and on roles of chaperones in assisting proteolysis of other client proteins).