SUMMARY We report a novel mechanism of ribonucleoprotein (RNP) nucleocytoplasmic export by nuclear envelope budding. During development of Drosophila synapses, a fragment of the Wnt-1 receptor, DFrizzled2, is imported into postsynaptic nuclei where it forms prominent foci. We now show these foci to be composed of large RNP granules harboring synaptic protein transcripts. These RNPs exit the nucleus via a budding mechanism akin to nuclear egress of Herpes-type viruses, a process previously thought to be exclusive to these viruses. During this mechanism, RNP granules bud into the space between the inner and the outer nuclear membranes (the perinuclear space), in a manner dependent on Lamin C, a nuclear protein linked to muscular dystrophies. Like herpes virus nuclear egress, this process requires protein kinase C, which is known to disrupt the lamin through phosphorylation. We suggest that nuclear budding is an endogenous nuclear export pathway for large RNP granules.

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production.Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNAinitiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.gene silencing | microRNA | transacting siRNA S mall silencing RNAs (sRNAs) in plants and animals, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in the development and the response to pathogens and stresses. These RNAs are also valuable tools in functional genomics and biotechnology. The sRNAs associate with ARGONAUTE (AGO) and other proteins in silencing effector complexes, and they bind to a target nucleic acid via Watson-Crick base pairing. In most instances, the silencing is a direct consequence of this interaction, and the AGO effector mediates RNA-mediated DNA or histone methylation, endonucleolytic RNA cleavage, or translational inhibition. In a few instances, an sRNA interaction also triggers the production of secondary siRNAs. The targeted RNA is converted into double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASEs (RDRs), which is then cleaved into the secondary siRNAs by DICER-LIKE (DCL) nucleases (1). Several proteins are known to be required for this process, but until now, the reason why most sRNA interactions do not result in secondary siRNA production was unclear.The transacting siRNA (tasiRNA) pathway in plants involves secondary siRNA production (2). Noncoding transcripts encoded by TAS1-4 genes serve as the precursors of tasiRNAs (3-5). After miRNA-directed cleavage, part of the remaining transcript is converted into dsRNA by RDR6. DCL4 then cleaves the dsRNA and generates tasiRNAs in a 21-nt phase relative to positions 10 and 11 of the miRNA that defines the site of targeted cleavage. TAS1 and TAS2 are targets of miR173, and their tasiRNAs in turn can target mRNAs for pentatricopeptide repeat (PPR) proteins. In one instance, a small sRNA cascade is initiated by miR173 (6, 7), because a TAS2-derived tasiRNA can itself initiate secondary siRNA production on several PPR mRNAs. The initiator of TAS3 tasiRNA is miR390 (3,8), and the TAS3 targets are AUXIN RESPONSE FACTOR mRNAs that influence the change from juvenile phase to adult phase, leaf morphology,...

Background: Release of microvesicles, including exosomes, is a novel mechanism of intercellular communication. At Drosophila synapses, the transmembrane Wnt-binding protein Evi/Wls is released in vesicles. Results: Evi-exosome release requires Rab11, Syntaxin 1A, and Myosin5. Conclusion: We established an in vivo system to elucidate the mechanisms of exosomal release. Significance: This is the first in vivo characterization of exosomal communication in the nervous system.

SUMMARY Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2-derived ta-siRNA instead of by microRNAs as was reported previously for TAS1a, -b, -c, TAS2, and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes.massively parallel signature sequencing ͉ TAS S mall regulatory RNAs modulate transcriptional gene silencing (TGS), mRNA degradation (post-TGS; PTGS) and translational repression in a wide spectrum of organisms. These small regulatory RNAs include microRNAs (miRNAs), heterochromatic siRNAs (hc-siRNAs), repeat-associated siRNAs (ra-siRNAs), natural sense-antisense transcript siRNAs (1), trans-acting siRNAs (ta-siRNAs) (2, 3), and the recently identified Piwi-interacting RNAs (4).In Arabidopsis, miRNAs are processed from hairpin precursors to play important roles in development and stress responses by either targeted cleavage of mRNA or translational repression (for review see ref. 5). The biogenesis of miRNAs requires a specific RNase III enzyme, DICER-LIKE protein 1 (DCL1) (5). Arabidopsis hcsiRNAs or ra-siRNAs trigger DNA methylation and histone modification and are thus involved in the assembly of heterochromatin and the control of transposon movement. hc-siRNAs or ra-siRNAs are usually derived from genomic repeats or transposons, a process requiring DICER-LIKE 3 (DCL3) and a specific RNA-dependent RNA polymerase, RDR2 (for reviews, see refs. 6 and 7).The identification of ta-siRNAs in Arabidopsis bridged the miRNA and siRNA pathways previously considered independent (2, 3, 8-10). ta-siRNAs clustered in 21-nt increments in both sense and antisense strands of several noncoding TAS transcripts were first identified from the study of two genes involved in PTGS, RDR6 and suppressor of gene silencing 3 (SGS3) (3, 8). Interestingly, the production of phased ta-siRNA is initially triggered by the targeted cleavage of primary TAS transcripts by miRNAs (2). After cleavage, the 5Ј or 3Ј cleavage products are converted into dsRNA with the assistanc...