Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex resembling enhanced GFP (EGFP), termed Spinach, emits a green fluorescence comparable in brightness to fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.

Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning, and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily acts to repel axons from inappropriate targets, induces cytoskeletal rearrangements that results in growth cone collapse 1 . These effects require intra-axonal mRNA translation. Here we show that transcripts for RhoA, a small GTPase that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3'UTR. Sema3A induces intra-axonal translation of RhoA mRNA and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a novel mechanism for the regulation of RhoA signaling.Studies using Xenopus retinal axons demonstrated that cytoskeletal regulation of the growth cone by Sema3A requires intra-axonal, or "local", mRNA translation 2 . Sema3A treatment results in increased protein synthesis in growth cones as evidenced by metabolic labeling experiments and by phosphorylation of elongation factor 4E-BP1 2 . These effects occur within minutes of Sema3A application 2 . Furthermore, Sema3A-mediated growth cone collapse is blocked by ribosomal inhibitors. The mRNA translation that is required for Sema3A-mediated growth cone collapse occurs in the axon, as both Sema3A-induced collapse and inhibition of this collapse by ribosomal inhibitors is preserved in axons that are severed from their cell bodies 2 .To determine if intra-axonal mRNA translation is required for Sema3A signaling in mammalian neurons, we examined Sema3A-mediated growth cone collapse in embryonic rat dorsal root ganglia (DRG) explant cultures 3 -5 . To eliminate the possibility that the effects of Sema3A were mediated through somatic translation, axons were severed from their cell bodies 2 ( Supplementary Fig. 1a). Treatment of severed axons with Sema3A for 60 min resulted in an increase in collapsed growth cones from 17 ± 1.3% to 75 ± 2.8 % ( Supplementary Fig. 1b, c, g). This effect was blocked by pretreatment of axons with either cycloheximide or anisomycin ( Supplementary Fig. 1d-f), both of which are ribosomal inhibitors. Pretreatment of these cultures with rapamycin, an inhibitor of cap-dependent translation 6 , also blocked Sema3A-mediated growth cone collapse. Together, these data indicate that the requirement forCorrespondence and requests for materials should be addressed to S.R.J. (e-mail:srj2003@med.cornell.edu).. * These authors contributed equally to this work.Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Competing interests statementThe authors declare that they have no competing financial interests. Supplementary ...

Summary The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA binding protein SAM68 which associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Growth cones at the tips of nascent and regenerating axons direct axon elongation. Netrin-1, a secreted molecule that promotes axon outgrowth and regulates axon pathfinding, elevates cyclic AMP (cAMP) levels in growth cones and regulates growth cone morphology and axonal outgrowth. These morphological effects depend on the intracellular levels of cAMP. However, the specific pathways that regulate cAMP levels in response to netrin-1 signaling are unclear. Here we show that 'soluble' adenylyl cyclase (sAC), an atypical calcium-regulated cAMP-generating enzyme previously implicated in sperm maturation, is expressed in developing rat axons and generates cAMP in response to netrin-1. Overexpression of sAC results in axonal outgrowth and growth cone elaboration, whereas inhibition of sAC blocks netrin-1-induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC-generated cAMP, and identify a fundamental role for sAC in axonal development.

BackgroundTo better understand potential transmission risks from contact with the body fluids of children, we monitored the presence and amount of CMV shedding over time in healthy CMV-seropositive children.MethodsThrough screening we identified 36 children from the Atlanta, Georgia area who were CMV-seropositive, including 23 who were shedding CMV at the time of screening. Each child received 12 weekly in-home visits at which field workers collected saliva and urine. During the final two weeks, parents also collected saliva and urine daily.ResultsPrevalence of shedding was highly correlated with initial shedding status: children shedding at the screening visit had CMV DNA in 84% of follow-up saliva specimens (455/543) and 28% of follow-up urine specimens (151/539); those not shedding at the screening visit had CMV DNA in 16% of follow-up saliva specimens (47/303) and 5% of follow-up urine specimens (16/305). Among positive specimens we found median viral loads of 82,900 copies/mL in saliva and 34,730 copies/mL in urine (P = 0.01), while the viral load for the 75th percentile was nearly 1.5 million copies/mL for saliva compared to 86,800 copies/mL for urine. Younger age was significantly associated with higher viral loads, especially for saliva (P < 0.001). Shedding prevalence and viral loads were relatively stable over time. All children who were shedding at the screening visit were still shedding at least some days during weeks 11 and 12, and median and mean viral loads did not change substantially over time.ConclusionsHealthy CMV-seropositive children can shed CMV for months at high, relatively stable levels. These data suggest that behavioral prevention messages need to address transmission via both saliva and urine, but also need to be informed by the potentially higher risks posed by saliva and by exposures to younger children.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0569-1) contains supplementary material, which is available to authorized users.