Single-molecule imaging of microRNA-mediated gene silencing in cells.

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown.

Identification of an AGO (Argonaute) protein as a prey of TER94/VCP.

VCP (valosin containing protein) recognizes a wide variety of substrates and mediates their degradation via the ubiquitin-proteasome system and macroautophagy/autophagy. The substrate repertoire of VCP, however, is not fully understood. In our recent study, we found that TER94/VCP mediates autophagic degradation of an Argonaute subfamily protein (AGO1), which binds microRNAs (miRNAs) and silences the expression of thousands of target genes.

VCP Machinery Mediates Autophagic Degradation of Empty Argonaute.

The Argonaute subfamily of proteins (AGO) loads microRNAs (miRNAs) to form the effector complex that mediates target gene silencing. Empty AGO, but not miRNA-loaded AGO, is selectively degraded across species. We have reported that the degradation of empty AGO is part of a quality control pathway that eliminates dysfunctional AGO.

Iruka Eliminates Dysfunctional Argonaute by Selective Ubiquitination of Its Empty State.

MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear.

RISC assembly: Coordination between small RNAs and Argonaute proteins.

Non-coding RNAs generally form ribonucleoprotein (RNP) complexes with their partner proteins to exert their functions. Small RNAs, including microRNAs, small interfering RNAs, and PIWI-interacting RNAs, assemble with Argonaute (Ago) family proteins into the effector complex called RNA-induced silencing complex (RISC), which mediates sequence-specific target gene silencing. RISC assembly is not a simple binding between a small RNA and Ago; rather, it follows an ordered multi-step pathway that requires specific accessory factors.

Measurement of Rab35 activity with the GTP-Rab35 trapper RBD35.

Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35).

Rab35 promotes the recruitment of Rab8, Rab13 and Rab36 to recycling endosomes through MICAL-L1 during neurite outgrowth.

Small GTPase Rab35 is an important molecular switch for endocytic recycling that regulates various cellular processes, including cytokinesis, cell migration, and neurite outgrowth. We previously showed that active Rab35 promotes nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells by recruiting MICAL-L1, a multiple Rab-binding protein, to Arf6-positive recycling endosomes. However, the physiological significance of the multiple Rab-binding ability of MICAL-L1 during neurite outgrowth remained completely unknown.

Rab35 is translocated from Arf6-positive perinuclear recycling endosomes to neurite tips during neurite outgrowth.

Small GTPase Rab35 is a key regulator of neurite outgrowth, and its activation dramatically enhances nerve growth factor (NGF)-induced neurite outgrowth. We recently reported finding that Rab35 and its effector molecules recruit EHD1, a facilitator of vesicle formation, to Arf6-positive perinuclear recycling endosomes (hereafter simply referred to as recycling endosomes) in response to NGF stimulation. Although Rab35 is likely to promote the formation of transport vesicles from recycling endosomes that contributes to neurite outgrowth, the destination of the vesicles during neurite outgrowth remains unknown.

Arf6, Rab11 and transferrin receptor define distinct populations of recycling endosomes.

Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations.

Rab35 establishes the EHD1-association site by coordinating two distinct effectors during neurite outgrowth.

Endocytic recycling is a process in which molecules that have been internalized are recycled back to the plasma membrane, and although it is crucial for regulating various cellular events, the molecular nexus underlying this process remains poorly understood. Here we report a molecular link between two gatekeepers for endocytic recycling, the molecular switch Rab35 and the molecular scissors EHD1, that is mediated by two distinct Rab35 effectors during neurite outgrowth of PC12 cells. Rab35 forms a tripartite complex with MICAL-L1 and centaurin-β2/ACAP2 and recruits them to perinuclear Arf6-positive endosomes in response to nerve growth factor stimulation.

Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

Two small GTPases, Rab and Arf, are well-known molecular switches that function in diverse membrane-trafficking routes in a coordinated manner; however, very little is known about the direct crosstalk between Rab and Arf. Although Rab35 and Arf6 were independently reported to regulate the same cellular events, including endocytic recycling, phagocytosis, cytokinesis and neurite outgrowth, the molecular basis that links them remains largely unknown. Here we show that centaurin-β2 (also known as ACAP2) functions both as a Rab35 effector and as an Arf6-GTPase-activating protein (GAP) during neurite outgrowth of PC12 cells.

Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35.

The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.

Comprehensive screening for novel rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs.

The Rab family belongs to the Ras-like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S-transferase (GST) pull-down assay with 60 different mammalian Rabs and mass spectroscopic analysis.