The RNA-binding protein RBP33 dampens non-productive transcription in trypanosomes.

In-depth analysis of the transcriptomes of several model organisms has revealed that genomes are pervasively transcribed, giving rise to an abundance of non-canonical and mainly antisense RNA polymerase II-derived transcripts that are produced from almost any genomic context. Pervasive RNAs are degraded by surveillance mechanisms, but the repertoire of proteins that control the fate of these non-productive transcripts is still incomplete. Trypanosomes are single-celled eukaryotes that show constitutive RNA polymerase II transcription and in which initiation and termination of transcription occur at a limited number of sites per chromosome. It is not known whether pervasive transcription exists in organisms with unregulated RNA polymerase II activity, and which factors could be involved in the process. We show here that depletion of RBP33 results in overexpression of ∼40% of all annotated genes in the genome, with a marked accumulation of sense and antisense transcripts derived from silenced regions. RBP33 loss does not result in a significant increase in chromatin accessibility. Finally, we have found that transcripts that increase in abundance upon RBP33 knockdown are significantly more stable in RBP33-depleted trypanosomes, and that the exosome complex is responsible for their degradation. Our results provide strong evidence that RBP33 dampens non-productive transcription in trypanosomes.

Synergistic regulation of Rgs4 mRNA by HuR and miR-26/RISC in neurons.

The negative regulator of G-protein signalling 4 (Rgs4) is linked to several neurologic diseases, e.g. schizophrenia, addiction, seizure and pain perception. Consequently, Rgs4 expression is tightly regulated, resulting in high mRNA and protein turnover. The post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. Here, we show that in neurons the RBP HuR reduces endogenous Rgs4 expression by destabilizing Rgs4 mRNA. Interestingly, in smooth muscle cells, Rgs4 is stabilized by HuR, indicating tissue-dependent differences in HuR function. Using in vitro RNA-based pulldown experiments, we identify the functional AU-rich element (ARE) within the Rgs4 3'-UTR that is recognized and bound by HuR. Bioinformatic analysis uncovered that this ARE lies within a highly conserved area next to a miR-26 binding site. We find that the neuronal-enriched miR-26 negatively influences Rgs4 expression in neurons. Further, HuR and miR-26 act synergistically in fluorescent reporter assays. Together, our data suggest a regulatory mechanism, in which an RBP selectively destabilizes a target mRNA in cooperation with a miRNA and the RISC machinery.

RGS4 RNA Secondary Structure Mediates Staufen2 RNP Assembly in Neurons.

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3'-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.

A retained intron in the 3'-UTR of Calm3 mRNA mediates its Staufen2- and activity-dependent localization to neuronal dendrites.

Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 (Calm3L ) mRNA Calm3L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3L mRNA to distal dendrites.

CLIPing Staufen to secondary RNA structures: size and location matter!

hiCLIP (RNA hybrid and individual-nucleotide resolution ultraviolet cross-linking and immunoprecipitation), is a novel technique developed by Sugimoto et al. (2015). Here, the use of different adaptors permits a controlled ligation of the two strands of a RNA duplex allowing the identification of each arm in the duplex upon sequencing. The authors chose a notoriously difficult to study double-stranded RNA-binding protein (dsRBP) termed Staufen1, a mammalian homolog of Drosophila Staufen involved in mRNA localization and translational control. Using hiCLIP, they discovered a dominance of intramolecular RNA duplexes compared to the total RNA duplexes identified. Importantly, the authors discovered two different types of intramolecular duplexes in the cell: highly translated mRNAs with long-range duplexes in their 3'-UTRs and poorly translated mRNAs with duplexes in their coding region. In conclusion, the authors establish hiCLIP as an important novel technique for the identification of RNA secondary structures that serve as in vivo binding sites for dsRBPs.

A short RNA stem-loop is necessary and sufficient for repression of gene expression during early logarithmic phase in trypanosomes.

We have compared the transcriptomes of cultured procyclic Trypanosoma brucei cells in early and late logarithmic phases and found that ∼200 mRNAs were differentially regulated. In late log phase cells, the most upregulated mRNA encoded the nucleobase transporter NT8. The 3' untranslated region (UTR) of NT8 contains a short stem-loop cis-element that is necessary for the regulation of NT8 expression in response to external purine levels. When placed in the 3'-UTR of an unregulated transcript, the cis-element is sufficient to confer regulation in response to purines. To our knowledge, this is the first example of a discrete RNA element that can autonomously regulate gene expression in trypanosomes in response to an external factor and reveals an unprecedented purine-dependent signaling pathway that controls gene expression in eukaryotes.

Fear extinction is regulated by the activity of long noncoding RNAs at the synapse.

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.

Live cell imaging reveals 3'-UTR dependent mRNA sorting to synapses.

mRNA transport restricts translation to specific subcellular locations, which is the basis for many cellular functions. However, the precise process of mRNA sorting to synapses in neurons remains elusive. Here we use Rgs4 mRNA to investigate 3'-UTR-dependent transport by MS2 live-cell imaging. The majority of observed RNA granules display 3'-UTR independent bidirectional transport in dendrites. Importantly, the Rgs4 3'-UTR causes an anterograde transport bias, which requires the Staufen2 protein. Moreover, the 3'-UTR mediates dynamic, sustained mRNA recruitment to synapses. Visualization at high temporal resolution enables us to show mRNA patrolling dendrites, allowing transient interaction with multiple synapses, in agreement with the sushi-belt model. Modulation of neuronal activity by either chemical silencing or local glutamate uncaging regulates both the 3'-UTR-dependent transport bias and synaptic recruitment. This dynamic and reversible mRNA recruitment to active synapses would allow translation and synaptic remodeling in a spatially and temporally adaptive manner.

The alternative life of RNA-sequencing meets single molecule approaches.

The central dogma of RNA processing has started to totter. Single genes produce a variety of mRNA isoforms by mRNA modification, alternative polyadenylation (APA), and splicing. Different isoforms, even those that code for the identical protein, may differ in function or spatiotemporal expression. One option of how this can be achieved is by the selective recruitment of trans-acting factors to the 3'-untranslated region of a given isoform. Recent innovations in high-throughput RNA-sequencing methods allow deep insight into global RNA regulation, whereas novel imaging-based technologies enable researchers to explore single RNA molecules during different stages of development, in different tissues and different compartments of the cell. Resolving the dynamic function of ribonucleoprotein particles in splicing, APA, or RNA modification will enable us to understand their contribution to pathological conditions.

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA.

Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.

Forebrain-specific, conditional silencing of Staufen2 alters synaptic plasticity, learning, and memory in rats.

BACKGROUND: Dendritic messenger RNA (mRNA) localization and subsequent local translation in dendrites critically contributes to synaptic plasticity and learning and memory. Little is known, however, about the contribution of RNA-binding proteins (RBPs) to these processes in vivo. RESULTS: To delineate the role of the double-stranded RBP Staufen2 (Stau2), we generate a transgenic rat model, in which Stau2 expression is conditionally silenced by Cre-inducible expression of a microRNA (miRNA) targeting Stau2 mRNA in adult forebrain neurons. Known physiological mRNA targets for Stau2, such as RhoA, Complexin 1, and Rgs4 mRNAs, are found to be dysregulated in brains of Stau2-deficient rats. In vivo electrophysiological recordings reveal synaptic strengthening upon stimulation, showing a shift in the frequency-response function of hippocampal synaptic plasticity to favor long-term potentiation and impair long-term depression in Stau2-deficient rats. These observations are accompanied by deficits in hippocampal spatial working memory, spatial novelty detection, and in tasks investigating associative learning and memory. CONCLUSIONS: Together, these experiments reveal a critical contribution of Stau2 to various forms of synaptic plasticity including spatial working memory and cognitive management of new environmental information. These findings might contribute to the development of treatments for conditions associated with learning and memory deficits.

RNA Transport: From Head to Toe in Radial Glial Cells.

Intracellular mRNA localization critically contributes to proper brain development and function. A recent study demonstrates that mRNAs are actively transported in radial glial cells from the soma to the distal basal endfeet, where they are locally translated.

Meet the players: local translation at the synapse.

It is widely believed that activity-dependent synaptic plasticity is the basis for learning and memory. Both processes are dependent on new protein synthesis at the synapse. Here, we describe a mechanism how dendritic mRNAs are transported and subsequently translated at activated synapses. Furthermore, we present the players involved in the regulation of local dendritic translation upon neuronal stimulation and their molecular interplay that maintain local proteome homeostasis. Any dysregulation causes several types of neurological disorders including muscular atrophies, cancers, neuropathies, neurodegenerative, and cognitive disorders.

Depletion of the RNA-binding protein RBP33 results in increased expression of silenced RNA polymerase II transcripts in Trypanosoma brucei.

We have characterized the RNA-binding protein RBP33 in Trypanosoma brucei, and found that it localizes to the nucleus and is essential for viability. The subset of RNAs bound to RBP33 was determined by immunoprecipitation of ribonucleoprotein complexes followed by deep sequencing. Most RBP33-bound transcripts are predicted to be non-coding. Among these, over one-third are located close to the end of transcriptional units (TUs) or have an antisense orientation within a TU. Depletion of RBP33 resulted in an increase in the level of RNAs derived from regions that are normally silenced, such as strand-switch regions, retroposon and repeat sequences. Our work provides the first example of an RNA-binding protein involved in the regulation of gene silencing in trypanosomes.

Alterations in DRBD3 ribonucleoprotein complexes in response to stress in Trypanosoma brucei.

Regulation of RNA polymerase II transcription initiation is apparently absent in trypanosomes. Instead, these eukaryotes control gene expression mainly at the post-transcriptional level. Regulation is exerted through the action of numerous RNA-binding proteins that modulate mRNA processing, turnover, translation and localization. In this work we show that the RNA-binding protein DRBD3 resides in the cytoplasm, but localizes to the nucleus upon oxidative challenge and to stress granules under starvation conditions. DRBD3 associates with other proteins to form a complex, the composition of which is altered by cellular stress. Interestingly, target mRNAs remain bound to DRBD3 under stress conditions. Our results suggest that DRBD3 transports regulated mRNAs within the cell in the form of ribonucleoprotein complexes that are remodeled in response to environmental cues.

Posttranscriptional control and the role of RNA-binding proteins in gene regulation in trypanosomatid protozoan parasites.

Trypanosomatids are unicellular eukaryotes responsible for severe diseases in humans. They exhibit a number of remarkable biological phenomena, especially at the RNA level. During their life cycles, they alternate between a mammalian host and an insect vector and undergo profound biochemical and morphological transformations in order to adapt to the different environments they find within one or the other host species. These changes are orchestrated by specific gene expression programs. In contrast to other organisms, trypanosomatids do not regulate RNA polymerase II-dependent transcription initiation. Evidence so far indicates that the main control points in gene expression are mRNA degradation and translation. Recent studies have shown that RNA-binding proteins (RBPs) play a critical role in the developmental regulation of mRNA and protein abundance. RBPs seem to bind to specific subsets of mRNAs encoding functionally related proteins. These ribonucleoprotein complexes may represent posttranscriptional operons or regulons that are able to control the fate of multiple mRNAs simultaneously. We suggest that trypanosomatids transduce environmental signals into mRNA and protein abundance through posttranslational modification of RBPs.