An atypical RNA quadruplex marks RNAs as vectors for gene silencing.

The addition of poly(UG) ('pUG') repeats to 3' termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.

Engineering a conserved RNA regulatory protein repurposes its biological function in vivo.

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA.

The astrocyte brain fatty acid binding protein (Fabp7) has previously been shown to have a coordinated diurnal regulation of mRNA and protein throughout mouse brain, and an age-dependent decline in protein expression within synaptoneurosomal fractions. Mechanisms that control time-of-day changes in expression and trafficking Fabp7 to the perisynaptic process are not known. In this study, we confirmed an enrichment of Fabp7 mRNA and protein in the astrocytic perisynaptic compartment, and observed a diurnal change in the intracellular distribution of Fabp7 mRNA in molecular layers of hippocampus. Northern blotting revealed a coordinated time-of-day-dependent oscillation for the Fabp7 mRNA poly(A) tail throughout murine brain. Cytoplasmic polyadenylation element-binding protein 1 (CPEB1) regulates subcellular trafficking and translation of synaptic plasticity-related mRNAs. Here we show that Fabp7 mRNA coimmunoprecipitated with CPEB1 from primary mouse astrocyte extracts, and its 3'UTR contains phylogenetically conserved cytoplasmic polyadenylation elements (CPEs) capable of regulating translation of reporter mRNAs during Xenopus oocyte maturation. Given that Fabp7 expression is confined to astrocytes and neural progenitors in adult mouse brain, the synchronized cycling pattern of Fabp7 mRNA is a novel discovery among known CPE-regulated transcripts. These results implicate circadian, sleep, and/or metabolic control of CPEB-mediated subcellular trafficking and localized translation of Fabp7 mRNA in the tripartite synapse of mammalian brain.

Tethered function assays: an adaptable approach to study RNA regulatory proteins.

Proteins and protein complexes that regulate mRNA metabolism must possess two activities. They bind the mRNA, and then elicit some function, that is, regulate mRNA splicing, transport, localization, translation, or stability. These two activities can often reside in different proteins in a complex, or in different regions of a single polypeptide. Much can be learned about the function of the protein or complex once it is stripped of the constraints imposed by RNA binding. With this in mind, we developed a "tethered function" assay, in which the mRNA regulatory protein is brought to the 3' UTR of an mRNA reporter through a heterologous RNA-protein interaction. In this manner, the functional activity of the protein can be studied independent of its intrinsic ability to recognize and bind to RNA. This simple assay has proven useful in dissecting numerous proteins involved in posttranscriptional regulation. We discuss the basic assay, consider technical issues, and present case studies that exemplify the strengths and limitations of the approach.