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Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules that are involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are localized to the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq, we identified a specific set of lncRNAs that accumulate in the synaptic compartment within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these was a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single-molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3BP2 and CAPRIN1, regulates the activity-dependent trafficking and clustering of RNA granules. In addition, we found that cell-type-specific, activity-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and RNA condensates in the synaptic compartment.

The RNA modification N6-methyladenosine (m6A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6A RNA-sequencing, we have discovered a distinct population of learning-related m6A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6A readers in the mPFC of male C57/BL6 mice, with m6A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m6A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.SIGNIFICANCE STATEMENT We have discovered that learning-induced m6A-modified RNA (including the long noncoding RNA, Malat1) accumulates in the synaptic compartment. We have identified several new m6A readers that are associated with fear extinction learning and demonstrate a causal relationship between m6A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m6A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.

The RNA modification N6 -methyladenosine (m6 A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6 A RNA-sequencing, we have discovered a distinct population of learning-related m6 A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6 A readers in the mPFC of male C57/BL6 mice, with m6 A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6 A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings high-light the critical role of m6 A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6 A readers in the synaptic compartment. [ABSTRACT FROM AUTHOR]

A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (~100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ~10 molecules/cell/min, leading at 5-10 min to a transient peak of ~150 phospho-ERK (pERK) molecules and ~50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome. [ABSTRACT FROM AUTHOR]

The RNA modification N 6 -methyladenosine (m 6 A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m 6 A RNA-sequencing, we have discovered a distinct population of learning-related m 6 A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 ( Malat1 ). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m 6 A readers in the mPFC of male C57/BL6 mice, with m 6 A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m 6 A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m 6 A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m 6 A readers in the synaptic compartment. SIGNIFICANCE STATEMENT We have discovered that learning-induced m 6 A-modified RNA (including the long noncoding RNA, Malat1 ) accumulates in the synaptic compartment. We have identified several new m 6 A readers that are associated with fear extinction learning and demonstrate a causal relationship between m 6 A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m 6 A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m 6 A readers in the synaptic compartment., (Copyright © 2023 the authors.)

Posttranscriptional regulation of RNA has emerged as an important regulator of genetic information flow in eukaryotic systems. In particular, chemical modifications of RNA have recently been established as key regulatory marks that affect the lifetime, location, trafficking, and function of messenger RNA (mRNA). In mammalian systems, N 6 -methyladenosine (m 6 A) is the most prevalent mRNA modification, and the writer, eraser, and reader proteins that install, remove, or recognize m 6 A have been rapidly uncovered and studied at the whole cell level. Understanding the effects of specific m 6 A modifications and their regulation at the single transcript level is the key next step to understanding the mechanism and consequences of epitranscriptomic regulation. We recently developed programmable m 6 A reader proteins to study the effects of epitranscriptomic regulatory factors at individual RNA transcripts. In this chapter, we discuss the application of targeted m 6 A readers to study RNA regulation at single endogenous sites. We briefly introduce what is currently known about the N 6 -methyltranscriptome and the Cas13 RNA-targeting family of proteins before detailing our protocol to study RNA modifications with targeted reader proteins., (© 2019 Elsevier Inc. All rights reserved.)

Post-transcriptional gene expression regulation of RNA has emerged as a key factor that controls mammalian protein production. RNA trafficking, translation efficiency, and stability are all controlled at the transcript level. For example, in addition to the commonly known processing steps of capping, splicing, and polyadenylation, RNA can be chemically modified. In eukaryotes, N 6 -methyladenosine (m 6 A) is the most prevalent mRNA modification. While the writers, erasers, and readers for m 6 A are rapidly being uncovered and studied at the whole-cell level, their competitive interplay to regulate methylated RNA transcripts has yet to be elucidated. To address this limitation, we report the development of programmable dPspCas13b-m 6 A reader proteins to investigate the regulatory effects of specific readers on single transcripts in live cells. We fused the two most well-characterized m 6 A reader proteins, YTHDF1 and YTHDF2, to a catalytically inactive PspCas13b protein, which can target the reader to a specific RNA of interest using guide RNA (gRNA) complementarity. We then demonstrate that the fused reader proteins each retain their reported functional role on a reporter construct: YTHDF2 induces degradation and YTHDF1 enhances translation. Finally, we show that the system can target endogenous mRNA transcripts within cells, using YTHDF2 as an exemplar, where we found tethering with YTHDF2 leads to decay of the target transcript. The development of dCas13b-based tools to study the regulation of endogenous RNAs will dramatically enhance our understanding of how RNA regulation occurs at the single RNA level. Additionally, our new tools, which permit transcript-specific mediated decay or enhanced protein production, will find utility in synthetic biology applications aimed at controlling genetic information flow at the RNA level.