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Fluorescence In Situ Hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. Here, we present an improved method for FISH probe production that yields high purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, Amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The Degree Of Labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO and Alexa dyes) was mostly greater than 90%, comparable to commercial probes. The ease and low cost of production allowed generation of probe-sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs forPolr2a(RNA polymerase II subunit 2a) andGapdh, and of the long noncoding RNAsMalat1andNeat1. Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in theGabbr1andNoc2ltranscripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

SUMMARYTo globally assess the distribution and processing of gene transcripts across subcellular compartments, we developed extensive RNA-seq datasets of both polyA+ and total RNA from chromatin, nucleoplasm and cytoplasm of mouse ESC, neuronal progenitors, and neurons. We identified protein-coding genes whose polyadenylated transcripts were more abundant in chromatin than cytoplasm. We defined introns exhibiting cotranscriptional splicing, complete intron retention in cytoplasmic RNA, and many introns retained in nucleoplasmic and chromatin RNA but absent from cytoplasmic RNA, including new introns controlled during neuronal development. In particular, we found that polyadenylated Gabbr1 transcripts are expressed in mESC but remain sequestered on chromatin until neuronal differentiation when they are processed and released to the cytoplasm. This developmental regulation of splicing and chromatin association demonstrates that the abundance of polyadenylated RNA is not always an indicator of functional gene expression. Our datasets provide a rich resource for analyzing many other aspects of mRNA maturation.

MicroRNA-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains non-neuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that pri-miR-124-1 is expressed in mouse embryonic stem cells (mESCs) where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem-loop to inhibit mature miR-124 expression in vivo, and DROSHA cleavage of pri-miR-124-1 in vitro. This new function for PTBP1 in repressing miR-124 biogenesis adds an additional regulatory loop to the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often modeled between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct de-repression of miR-124, in addition to indirect effects previously described.

Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.

The metabotropic glutamate receptors (mGluRs) are G-proteincoupled receptors whose activation modulates neural activity via their linkage to multiple second-messenger systems (Pin and Duvoisin 1995; Anwyl 1999). Eight mGluRs have been cloned and classified into three groups based on their sequence homologies, signal transduction mechanisms, and agonist pharmacology (Nakanishi 1992; Pin and Duvoisin 1995). Group I (mGluR1 and mGluR5) receptors stimulate phosphatidylinositol hydrolysis, whereas group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) receptors are negatively coupled to cAMP production. The group II mGluRs are implicated as negative feedback autoreceptors to modulate neurotransmission by presynaptic mechanisms (Pin and Duvoisin 1995). In situ hybridization and immunohistochemical studies of rat brain have revealed that the intense mGluR II-like immunoreactivity was seen predominantly in the cortical and limbic regions including the amygdala (Neki et al. 1996; Schaffhauser et al. 1998), suggesting that these receptors may modulate higher CNS functions such as emotion and cognition. Indeed, mGluR II agonists have been shown to inhibit the release of glutamate in vivo (Battaglia et al. 1997) and exhibit potent anxiolytic effects in rats (Helton et al. 1998). These compounds also ameliorated the symptoms of nicotine withdrawal (Helton et al. 1997) and lactate-induced panic responses (Shekhar and Keim 2000). Moreover, mice lacking mGluR II display impaired hippocampal mossy fiber long-term depression (LTD) (Yokoi et al. 1996). In external capsule (EC)-basolateral amygdala synapses, it has been shown that low-frequency stimulation (LFS) induced long-term potentiation (LTP), whereas brief high-frequency stimulation (HFS) evoked short-term potentiation that generally decayed within 10 min. When LFS was applied after recovery from the short-term potentiating effect of HFS, there was an initial transient enhancement followed by LTD. The switch in the direction of the enduring change in synaptic strength required activation of mGluR II (Li et al. 1998). LTP of synaptic responses could be reversed by LFS of afferent fibers, a phenomenon termed “depotentiation” (Barrionuevo et al. 1980; Staubli and Chun 1996). Previous studies indicated that LTP of synapses from auditory thalamus and cortex to the lateral amygdala (LA) underlay the encoding of fear memory (McKernan and Shinnick-Gallagher 1997; Rogan et al. 1997). Yet, it is not clear whether depotentiation contributes to behavioral extinction of fear memory. In the present study, we obtained evidence that activation of mGluR II induces depotentiation in rat amygdala slices. This chemical depotentiation is independent of NMDA receptor, L-type Ca++ channels, and calcineurin activity, but requires presynaptic activity and extracellular Ca++. By behavioral tests, we further demonstrate that administration of mGluR II agonists to the LA or basolateral amygdala (BLA) blocks the consolidation of fear memory measured with fear-potentiated startle.