Extracellular vesicles, especially the small exosome-like vesicles that originate from multivesicular endosomes, carry lipids, proteins, and RNA between cells. Among their most studied cargo are microRNAs, roughly 22-nucleotide RNA molecules that silence target genes by binding complementary messenger RNAs and recruiting the RNA-induced silencing complex. In the nervous system, microRNAs are critical regulators of local protein synthesis at sites far from the cell body, making them essential for building neuronal circuits and for synaptic plasticity. Because neurons depend on precise translational control within discrete compartments such as dendrites, the delivery of functional microRNAs between cells could be especially consequential. Previous work by the same team showed that neuronal small vesicles contribute to synapse formation downstream of BDNF-TrkB signaling, partly through the specific sorting and delivery of microRNA cargo to recipient neurites. But the question of how particular microRNAs are chosen for export has remained open.

Scientists knew that several RNA-binding proteins can escort microRNAs into vesicles. The protein hnRNPA2B1, for example, was one of the first shown to mediate microRNA secretion when sumoylated, preferentially binding microRNAs carrying a GGAG motif at their 3′ end, a sequence dubbed the EXO motif. Other proteins, including YBX-1 and Syncrip, also known as hnRNPQ, bind microRNAs through different sequence motifs and escort them into vesicles. Intracellular architecture matters too: membrane contact sites between the endoplasmic reticulum and endosomes, formed by the linker protein VAP-A, have been shown to drive the release of RNA-enriched vesicles, and liquid-liquid phase condensates containing RNA are thought to participate in sorting. What remained unclear was how these mechanisms are coupled, and how the vesicle-bound RNA-binding proteins actually receive their microRNA cargo.

The new study points to an unexpected answer: the microRNA production line itself. After precursor microRNAs are exported from the nucleus, the RNase III enzyme Dicer cleaves them into mature duplexes, which are then loaded onto Argonaute proteins to form the functional silencing complex. TRBP, a Dicer-interacting protein, promotes accurate cleavage by holding Dicer in a cleavage-competent open conformation and helps load the duplex onto Argonaute through a ternary assembly called the RISC-loading complex. This complex preferentially nucleates on the cytoplasmic surface of the endoplasmic reticulum. The researchers reasoned that if microRNA production happens at the ER, and vesicle sorting also involves ER-endosome contacts, the production machinery might serve as the sorting hub.

To test this, the team performed mass spectrometry on proteins pulled down with TRBP from primary neuronal lysates and compared the results with their previously published proteomics dataset of neuronal extracellular vesicles. A substantial fraction of proteins overlapped between the two datasets, and network analysis revealed enrichment of messenger RNA and microRNA binding proteins as well as cytoskeletal constituents. The researchers then validated interactions between TRBP and candidate cargo proteins, including YBX-1, hnRNPA2B1, and Syncrip, in neuroblastoma cells. Notably, the interaction between TRBP and hnRNPA2B1 disappeared when RNA was digested with RNase A, indicating it depends on RNA, whereas the Syncrip-TRBP interaction actually strengthened in the absence of RNA, suggesting a direct protein-protein contact. In primary cortical neurons, endogenous TRBP and Syncrip were confirmed to interact, and the two proteins partially co-localized near synaptophysin-positive synapses in the dendrites of mature hippocampal neurons.

Importantly, although Syncrip and Argonaute 2 were detected in the vesicle fraction, the core RISC-loading complex proteins Dicer, TRBP, and PACT were not secreted. This means TRBP stays behind inside the cell while handing off its cargo to proteins that do get exported. When the researchers knocked down TRBP in primary cortical neurons using siRNA and sequenced the small RNAs in cell lysates and purified vesicles, they found pronounced changes in vesicle microRNA content, with little correlation between the changes in vesicles and those in cell lysates. That lack of correlation is a key clue: it indicates that TRBP affects not simply how much microRNA is made, but which microRNAs are selectively sorted into vesicles. Gene ontology analysis of predicted targets pointed to processes related to synapse and dendrite development, synapse organization, and synaptic activity.

Motif enrichment analysis sharpened the picture. Among the microRNAs regulated by TRBP knockdown in vesicles, the most enriched six-nucleotide motif was GGC[A/U], the previously reported Syncrip-binding sequence, appearing in 19 percent of targets with 4.6-fold enrichment. This motif was not enriched in cell lysates, suggesting TRBP specifically promotes the sorting of Syncrip-binding microRNAs into vesicles. Additional motifs matching known vesicle-associated RNA-binding proteins, including hnRNPU, La protein, and Annexin A2, were also enriched. SynGO analysis of the predicted targets of TRBP-regulated, Syncrip-motif-containing microRNAs showed highly significant enrichment for synaptic localization and function. Among these microRNAs are miR-27b-3p, known to shape the presynaptic transcriptome, and miR-182-5p and miR-183-5p, members of a cluster implicated in long-term memory formation.

Using deletion mutants, the team mapped the interaction to the C-terminal domain of Syncrip: removing this region abolished TRBP binding, while removing the N-terminal domain did not. RNA immunoprecipitation experiments showed that the C-terminal mutant also lost its preferential binding to miR-27b-3p, a microRNA containing the Syncrip-like motif, while binding of a microRNA with a different motif was unaffected and a motif-free microRNA did not bind at all. The C-terminal mutant also failed to interact with Argonaute 2 and rarely formed the punctate RNA granules that wild-type Syncrip occupies, granules that overlap with P-bodies, the cytoplasmic sites of microRNA-mediated repression. Meanwhile, the TRBP orthologue PACT bound more strongly to the C-terminal mutant, hinting at an alternative, possibly siRNA-related sorting route that the authors say requires further verification.

The spatial control of this pathway proved to be just as striking as its biochemistry. Because the TRBP-containing RISC-loading complex assembles on the ER surface, the researchers manipulated membrane contact sites between the ER and endosomes using engineered forms of the linker proteins Protrudin and ORP1L. Blocking ER-endosome contact sites increased the recruitment of Syncrip to ER-associated TRBP, confirmed by proximity ligation assay in hippocampal neurons, and increased the abundance of Syncrip and Argonaute in vesicles immunocaptured with antibodies against CD63, CD81, and CD9. Conversely, increasing endosome contact sites shifted Syncrip and Argonaute 2 granules toward late endosomes. The picture that emerges is of a dynamic gatekeeping system in which the flickering contacts between ER and endosomes determine whether Syncrip binds the microRNA factory and loads de novo produced microRNAs, or lingers on endosomes and RNA granules instead.

The functional payoff came from experiments on synapse formation. Overexpressing wild-type Syncrip increased the density of synapses, measured by Synapsin and Homer co-localization, in donor neurons, whereas the TRBP-binding-deficient C-terminal mutant did not. Remarkably, the effect reversed in recipient neurons: vesicles collected from neurons expressing either Syncrip mutant increased synapse density in the cells that received them, while vesicles from wild-type-expressing donors did not. Dendrite complexity was unchanged in all conditions, arguing against a general trophic effect and pointing specifically at synapse regulation. When the researchers depleted RNA from vesicles using RNase A delivered through mild saponin permeabilization, the synapse-promoting effect of vesicles from C-terminal mutant-expressing neurons was abolished, confirming that RNA cargo drives that phenotype. Vesicles from N-terminal mutant neurons retained their effect even after RNA depletion, and even protein fractions depleted of vesicles influenced recipient synapses, suggesting the N-terminus of Syncrip shapes additional, non-RNA cargo. The authors propose that ER-associated TRBP acts as a gatekeeper for synaptic microRNAs: retaining them strengthens the donor neuron’s synapses, while releasing them in vesicles allows those same molecules to tune synapse formation in neighboring cells, a competitive mechanism that could help fine-tune microcircuits during development. Given that impaired ER-endosome contact sites have been implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases, and that vesicles are increasingly pursued as vehicles for RNA-based therapies, understanding this sorting hub may ultimately inform the engineering of vesicles with precisely controlled microRNA payloads.

Subject of Research: Mechanisms of microRNA sorting into neuronal extracellular vesicles during synaptogenesis

Article Title: The Cytoplasmic MicroRNA‐Production Complex Regulates MicroRNA Sorting to Extracellular Vesicles During Synaptogenesis

Article References: Zeng, Y., Luhmann, S., Saha, A., Orlando, M., Sami, H., Glatter, T., & Antoniou, A. (2026). The Cytoplasmic MicroRNA‐Production Complex Regulates MicroRNA Sorting to Extracellular Vesicles During Synaptogenesis. Advanced Science, Article e78067. https://doi.org/10.1002/advs.78067

Image Credits: AI Generated

DOI: 10.1002/advs.78067

Keywords: extracellular vesicles, microRNA, TRBP, Syncrip, synaptogenesis, endoplasmic reticulum, membrane contact sites, RISC-loading complex, Argonaute, RNA-binding proteins, neurons, RNA sorting