Project description: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). 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 AGO1, and with membrane-bound polysomes is increased in farnesyl transferase and farnesylationdeficient J2/J3 mutants. Third, silencing by non-cell autonomously acting short interfering RNAs (siRNAs) 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.
Project description:The endoplasmic reticulum (ER) was implicated as the site of microRNA (miRNA)-mediated translation repression in plants. Here, we examined the ER- and rough ER-associated small RNAome, transcriptome and translatome. We found that almost all cellular transcripts were present on membrane-bound polysomes (MBPs), and miRNAs were enriched in membranes and on MBPs. miRNAs were recruited to membranes by their effector protein AGO1, whereas AGO1 associated with membranes and, partly ribosomes, in an RNA-independent manner. Most strikingly, 22 ntmiRNA isoformsand a set of 22 ntsiRNAs, were enriched in the membrane fraction and onMBPs, where they trigger phasedsecondary siRNAproduction from target transcripts. Loss of membrane association of the 22 nt small RNAs resulted in the loss of phasing in secondary siRNA production.These findings point to the ER as a hub that hosts and organizes endogenous small RNAs in plants.
Project description:The endoplasmic reticulum (ER) was implicated as the site of microRNA (miRNA)-mediated translation repression in plants. Here, we examined the ER- and rough ER-associated small RNAome, transcriptome and translatome. We found that almost all cellular transcripts were present on membrane-bound polysomes (MBPs), and miRNAs were enriched in membranes and on MBPs. miRNAs were recruited to membranes by their effector protein AGO1, whereas AGO1 associated with membranes and, partly ribosomes, in an RNA-independent manner. Most strikingly, 22 ntmiRNA isoformsand a set of 22 ntsiRNAs, were enriched in the membrane fraction and onMBPs, where they trigger phasedsecondary siRNAproduction from target transcripts. Loss of membrane association of the 22 nt small RNAs resulted in the loss of phasing in secondary siRNA production.These findings point to the ER as a hub that hosts and organizes endogenous small RNAs in plants.
Project description:Endoplasmic reticulum (ER) was implicated as the site of microRNA (miRNA)-mediated translational repression in plants. Here, we examined the ER- and rough ER- associated small RNAome, transcriptome and translatome. We found that nearly all cellular transcripts were present on membrane-bound polysomes (MBPs), and miRNAs and a small set of endogenous siRNAs were particularly enriched on MBPs. The MBP- enriched miRNAs and siRNAs associated with, and were recruited to membranes by, their effector protein ARGONAUTE1 (AGO1). AGO1 associated with ER in a partly RNA-independent manner. Reduced membrane association of 22-nt miRNAs, which trigger the biogenesis of phased, secondary siRNAs (phasiRNAs) from their target transcripts, was accompanied by decreased production or loss of phasing of phasiRNAs. The phasiRNA precursor transcripts, previously thought to be noncoding, were associated with MBPs in a manner that supported phasiRNA production. These findings point to the ER as a hub that hosts and organizes endogenous small RNAs in plants.
Project description:The cytoplasm contains membrane-bound organelles and membraneless compartments. The TIS granule network is formed through assembly of the RNA-binding protein TIS11B together with its bound mRNAs and associates with a portion of the rough endoplasmic reticulum (ER) – one of the major sites of protein synthesis. Subcellular mRNA localization is widespread in polarized cells. However, it is largely unknown if mRNA localization is also widespread in non-polarized cells and if the location of protein synthesis has biological consequences. To identify compartment-specific cellular functions, we used fluorescent particle sorting to isolate TIS granules, the ER surface, and the cytosol and determined their mRNA contents.
Project description:The cytoplasm contains membrane-bound organelles and membraneless compartments. The TIS granule network is formed through assembly of the RNA-binding protein TIS11B together with its bound mRNAs and associates with a portion of the rough endoplasmic reticulum (ER) – one of the major sites of protein synthesis. Subcellular mRNA localization is widespread in polarized cells. However, it is largely unknown if mRNA localization is also widespread in non-polarized cells and if the location of protein synthesis has biological consequences. To identify compartment-specific cellular functions, we used fluorescent particle sorting to isolate TIS granules, the ER surface, and the cytosol and determined their mRNA contents.
Project description:Purpose: We investigated Nr4a effector genes following a hippocampus dependent learning task using a dominant mouse model of Nr4a. Method: We performed total RNA sequencing after training for hippocampus-dependent memory from Nr4ADN and control littermates Results: Using an unbiased analysis of gene expression after training for hippocampus-dependent memory, we found that Nr4a transcription factors regulate expression of specific effector genes encoding endoplasmic reticulum (ER) chaperones that drive protein folding, enabling membrane proteins to more efficiently traffic to the cell surface. Conclusion: This study identified the effector genes of Nr4a transcription factors involved in learning and memory.
Project description:Mammalian ATF6M-NM-1/M-NM-2 are membrane-bound transcription factors which are activated by endoplasmic reticulum (ER) stress-induced proteolysis to upregulate various ER quality control proteins to maintain the homeostasis of the ER. ATF6M-NM-1- and ATF6M-NM-2-single knockout mice develop normally but ATF6M-NM-1/M-NM-2-double knockout causes embryonic lethality, the reason for which remains unknown. Here, we showed that medaka fish exhibits the same phenotype regarding the effects of deleting ATF6M-NM-1, ATF6M-NM-2, and both. Analyses revealed that ER stress occurred physiologically during early embryonic development, particularly in the brain, otic vesicle and notochord. The absence of transcriptional induction of ER chaperones in ATF6M-NM-1/M-NM-2-double knockout blocked notochord development, which was partially rescued by microinjection-mediated overexpression of the major ER chaperone BiP. Thus, ATF6M-NM-1/M-NM-2-mediated adjustment of chaperones to the increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis. Gene expression profile at stage 24 of DKO fish (ATF6a -/-b-/-) and control fish (ATF6a+/- b+/-) was determined. Two independent experiments were performed.