Overlapping Activities of ELAV/Hu Family RNA Binding Proteins Specify the Extended Neuronal 3' UTR Landscape in Drosophila.

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.

ELAV and FNE Determine Neuronal Transcript Signatures through EXon-Activated Rescue.

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.

The RNA-binding protein HuR is essential for the B cell antibody response.

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

RNA metabolism. Promoters drive alternative polyadenylation.

The RNA-binding protein Elav is recruited in the fruit fly to promoter regions, possibly by paused RNA polymerase II, to promote alternative polyadenylation.

The formation of HuR/YB1 complex is required for the stabilization of target mRNA to promote myogenesis.

mRNA stability is the mechanism by which cells protect transcripts allowing their expression to execute various functions that affect cell metabolism and fate. It is well-established that RNA binding proteins (RBPs) such as HuR use their ability to stabilize mRNA targets to modulate vital processes such as muscle fiber formation (myogenesis). However, the machinery and the mechanisms regulating mRNA stabilization are still elusive. Here, we identified Y-Box binding protein 1 (YB1) as an indispensable HuR binding partner for mRNA stabilization and promotion of myogenesis. Both HuR and YB1 bind to 409 common mRNA targets, 147 of which contain a U-rich consensus motif in their 3' untranslated region (3'UTR) that can also be found in mRNA targets in other cell systems. YB1 and HuR form a heterodimer that associates with the U-rich consensus motif to stabilize key promyogenic mRNAs. The formation of this complex involves a small domain in HuR (227-234) that if mutated prevents HuR from reestablishing myogenesis in siHuR-treated muscle cells. Together our data uncover that YB1 is a key player in HuR-mediated stabilization of pro-myogenic mRNAs and provide the first indication that the mRNA stability mechanism is as complex as other key cellular processes such as mRNA decay and translation.

A genome-wide CRISPR screen identifies HuR as a regulator of apoptosis induced by dsRNA and virus.

We performed an unbiased whole-genome CRISPR/Cas9 screen in A549 cells to identify potential regulators involved in cell death triggered by double-stranded RNA (dsRNA). Of several top candidate genes, we identified the RNA-binding gene ELAV like protein 1 (256529), which encodes the protein Hu antigen R (HuR). Depletion of HuR led to less cell death induced by dsRNA. HuR is mainly involved in apoptosis, and all of its RNA recognition motifs are essential for its pro-apoptotic function. We further showed that the HuR depletion had no influence on the mRNA level of the anti-apoptotic gene BCL2, but instead that HuR downregulates BCL2 translation in a cap-independent way. Polysome fractionation studies showed that HuR retarded the BCL2 mRNA in the non-translating pool of polysomes. Moreover, protection from dsRNA-induced apoptosis by HuR depletion required the presence of BCL2, indicating that the pro-apoptotic function of HuR is executed by suppressing BCL2. Consistent with this, HuR regulated apoptosis induced by infection of encephalomyocarditis or Semliki Forest virus. Collectively, our work identified a suite of proteins that regulate dsRNA-induced cell death, and elucidated the mechanism by which HuR acts as a pro-apoptotic factor.

Different Genetic Signatures of Small-Cell Lung Cancer Characterize Anti-GABAB R and Anti-Hu Paraneoplastic Neurological Syndromes.

OBJECTIVE: Small-cell lung cancer (SCLC) is the malignancy most frequently associated with paraneoplastic neurological syndromes (PNS) and can trigger different antibody responses against intracellular (Hu) or neuronal surface (GABAB R) antigens. Our aim was to clarify whether the genomic and transcriptomic features of SCLC are different in patients with anti-GABAB R or anti-Hu PNS compared with SCLC without PNS. METHODS: A total of 76 SCLC tumor samples were collected: 34 anti-Hu, 14 anti-GABAB R, and 28 SCLC without PNS. The study consisted of 4 steps: (1) pathological confirmation; (2) next generation sequencing using a panel of 98 genes, including those encoding the autoantibodies targets ELAVL1-4, GABBR1-2, and KCTD16; (3) genome-wide copy number variation (CNV); and (4) whole-transcriptome RNA sequencing. RESULTS: CNV analysis revealed that patients with anti-GABAB R PNS commonly have a gain in chromosome 5q, which contains KCTD16, whereas anti-Hu and control patients often harbor a loss. No significantly different number of mutations regarding any onconeural genes was observed. Conversely, the transcriptomic profile of SCLC was different, and the differentially expressed genes allowed effective clustering of the samples into 3 groups, reflecting the antibody-based classification, with an overexpression of KCTD16 specific to anti-GABAB R PNS. Pathway analysis revealed that tumors of patients with anti-GABAB R encephalitis were enriched in B-cell signatures, as opposed to those of patients with anti-Hu, in which T-cell- and interferon-γ-related signatures were overexpressed. INTERPRETATION: SCLC genetic and transcriptomic features differentiate anti-GABAB R, anti-Hu, and non-PNS tumors. The role of KCTD16 appears to be pivotal in the tumor immune tolerance breakdown of anti-GABAB R PNS. ANN NEUROL 2023;94:1102-1115.

Cell-Penetrating Peptide TAT-HuR-HNS3 Suppresses Proinflammatory Gene Expression via Competitively Blocking Interaction of HuR with Its Partners.

Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinflammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inflammation-related diseases.

Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference.

Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. Here, we investigate the iab-8 ncRNA from the Drosophila Bithorax Complex and show that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, we find that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote.

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing.

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons.

Transcription initiation determines its end.

A new study published in this issue of Molecular Cell (Oktaba et al., 2015) suggests widespread involvement of promoters in the regulation of alternative cleavage and polyadenylation of mRNAs in Drosophila neurons.

ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system.

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. Here, we present evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. Our study provides evidence for a regulatory link between promoter-proximal pausing and APA.

Short- and Long-Term Regulation of HuD: A Molecular Switch Mediated by Folic Acid?

The RNA-binding protein HuD has been shown to play a crucial role in gene regulation in the nervous system and is involved in various neurological and psychiatric diseases. In this study, through the creation of an interaction network on HuD and its potential targets, we identified a strong association between HuD and several diseases of the nervous system. Specifically, we focused on the relationship between HuD and the brain-derived neurotrophic factor (BDNF), whose protein is implicated in several neuronal diseases and is involved in the regulation of neuronal development, survival, and function. To better investigate this relationship and given that we previously demonstrated that folic acid (FA) is able to directly bind HuD itself, we performed in vitro experiments in neuron-like human SH-SY5Y cells in the presence of FA, also known to be a pivotal environmental factor influencing the nervous system development. Our findings show that FA exposure results in a significant increase in both HuD and BDNF transcripts and proteins after 2 and 4 h of treatment, respectively. Similar data were obtained after 2 h of FA incubation followed by 2 h of washout. This increase was no longer detected upon 24 h of FA exposure, probably due to a signaling shutdown mechanism. Indeed, we observed that following 24 h of FA exposure HuD is methylated. These findings indicate that FA regulates BDNF expression via HuD and suggest that FA can behave as an epigenetic modulator of HuD in the nervous system acting via short- and long-term mechanisms. Finally, the present results also highlight the potential of BDNF as a therapeutic target for specific neurological and psychiatric diseases.

ELAV Proteins Bind and Stabilize C/EBP mRNA in the Induction of Long-Term Memory in Aplysia.

Long-term memory (LTM) formation is a critical survival process by which an animal retains information about prior experiences to guide future behavior. In the experimentally advantageous marine mollusk Aplysia, LTM for sensitization can be induced by the presentation of two aversive shocks to the animal's tail. Each of these training trials recruits distinct growth factor signaling systems that promote LTM formation. Specifically, whereas intact TrkB signaling during Trial 1 promotes an initial and transient increase of the immediate early gene apc/ebp mRNA, a prolonged increase in apc/ebp gene expression required for LTM formation requires the addition of TGFß signaling during Trial 2. Here we explored the molecular mechanisms by which Trial 2 achieves the essential prolonged gene expression of apc/ebp We find that this prolonged gene expression is not dependent on de novo transcription, but that apc/ebp mRNA synthesized by Trial 1 is post-transcriptionally stabilized by interacting with the RNA-binding protein ApELAV. This interaction is promoted by p38 MAPK activation initiated by TGFß. We further demonstrate that blocking the interaction of ApELAV with its target mRNA during Trial 2 blocks both the prolonged increase in apc/ebp gene expression and the behavioral induction of LTM. Collectively, our findings elucidate both when and how ELAV proteins are recruited for the stabilization of mRNA in LTM formation. Stabilization of a transiently expressed immediate early gene mRNA by a repeated training trial may therefore serve as a "filter" for learning, permitting only specific events to cause lasting transcriptional changes and behavioral LTM.SIGNIFICANCE STATEMENT: In the present paper, we significantly extend the general field of molecular processing in long-term memory (LTM) by describing a novel form of pretranslational processing required for LTM, which relies on the stabilization of a newly synthesized mRNA by a class of RNA binding proteins (ELAVs). There are now compelling data showing that important processing can occur after transcription of a gene, but before translation of the message into protein. Although the potential importance of ELAV proteins in LTM formation has previously been reported, the specific actions of ELAV proteins during LTM formation remained to be understood. Our new findings thus complement and extend this literature by demonstrating when and how this post-transcriptional gene regulation is mediated in the induction of LTM.

SRI-42127, a novel small molecule inhibitor of the RNA regulator HuR, potently attenuates glial activation in a model of lipopolysaccharide-induced neuroinflammation.

Glial activation with the production of pro-inflammatory mediators is a major driver of disease progression in neurological processes ranging from acute traumatic injury to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. Posttranscriptional regulation is a major gateway for glial activation as many mRNAs encoding pro-inflammatory mediators contain adenine- and uridine-rich elements (ARE) in the 3' untranslated region which govern their expression. We have previously shown that HuR, an RNA regulator that binds to AREs, plays a major positive role in regulating inflammatory cytokine production in glia. HuR is predominantly nuclear in localization but translocates to the cytoplasm to exert a positive regulatory effect on RNA stability and translational efficiency. Homodimerization of HuR is necessary for translocation and we have developed a small molecule inhibitor, SRI-42127, that blocks this process. Here we show that SRI-42127 suppressed HuR translocation in LPS-activated glia in vitro and in vivo and significantly attenuated the production of pro-inflammatory mediators including IL1ß, IL-6, TNF-α, iNOS, CXCL1, and CCL2. Cytokines typically associated with anti-inflammatory effects including TGF-ß1, IL-10, YM1, and Arg1 were either unaffected or minimally affected. SRI-42127 suppressed microglial activation in vivo and attenuated the recruitment/chemotaxis of neutrophils and monocytes. RNA kinetic studies and luciferase studies indicated that SRI-42127 has inhibitory effects both on mRNA stability and gene promoter activation. In summary, our findings underscore HuR's critical role in promoting glial activation and the potential for SRI-42127 and other HuR inhibitors for treating neurological diseases driven by this activation.

Inhibition of the DAPKs-L13a axis prevents a GAIT-like motif-mediated HuR insufficiency in melanoma cells.

We previously showed that the adaptive response of BRAFV600-mutated melanoma cells to BRAF inhibition emerges from a subpopulation of cells expressing an intermittent lower level of the mRNA-binding protein HuR. In this study, following initial overexpression experiments in which we confirm our previous results, we use wild-type and mutants HuR full-length mRNA constructs and in vivo-interacting assays and demonstrate that a highly conserved interferon-γ-activated inhibitor of translation (GAIT)-like motif located upstream of the GU-rich elements of HuR major polyadenylation site (PAS2), interacts with constituents of the GAIT complex and affects HuR post-transcriptional expression regulation. Knockdown of the ribosomal protein L13a or the inhibition of the DAPK1-ZIPK axis involved in L13a phosphorylation, reduces the proportion of HuRLow cells at steady-state and attenuates the adaptive response of the whole melanoma-cell population to BRAF inhibition. These results have further potential therapeutic implications for disease conditions associated with HuR insufficient expression.

A feedforward regulatory loop between HuR and the long noncoding RNA linc-MD1 controls early phases of myogenesis.

The muscle-specific long noncoding RNA linc-MD1 was shown to be expressed during early phases of muscle differentiation and to trigger the switch to later stages by acting as a sponge for miR-133 and miR-135. Notably, linc-MD1 is also the host transcript of miR-133b, and their biogenesis is mutually exclusive. Here, we describe that this alternative synthesis is controlled by the HuR protein, which favors linc-MD1 accumulation through its ability to bind linc-MD1 and repress Drosha cleavage. We show that HuR is under the repressive control of miR-133 and that the sponging activity of linc-MD1 consolidates HuR expression in a feedforward positive loop. Finally, we show that HuR also acts in the cytoplasm, reinforcing linc-MD1 sponge activity by cooperating for miRNA recruitment. An increase in miR-133 synthesis, mainly from the two unrelated miR-133a coding genomic loci, is likely to trigger the exit from this circuitry and progression to later differentiation stages.

Direct binding of Ataxin-2 to distinct elements in 3' UTRs promotes mRNA stability and protein expression.

It has been proposed that Ataxin-2, a member of the like-Sm (LSm) protein family, participates in the regulation of RNA metabolism through interaction with PABPC1. However, the exact biological mechanism and in vivo targets remain unknown. Here, we report that Ataxin-2 binds directly to RNAs in a PABPC1-independent manner. High-throughput sequencing of Ataxin-2-bound RNAs prepared by PAR-CLIP revealed that Ataxin-2 binds predominantly to uridine-rich elements, including well-characterized cis-regulatory AU-rich elements, in the 3' UTRs of target mRNAs. Gene expression analysis after Ataxin-2 depletion or overexpression revealed that Ataxin-2 stabilizes target mRNAs and increases the abundance of the corresponding proteins. A tethering assay demonstrated that Ataxin-2 elicits this effect by direct interaction with mRNAs. We also found that disease-associated polyglutamine expansion downregulates the physiological activity of Ataxin-2. These findings suggest that Ataxin-2 is an RNA-binding protein that targets cis-regulatory elements in 3' UTRs to stabilize a subset of mRNAs and increase protein expression.

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

The use of eye-movement recording in patients with anti-Hu antibody-associated paraneoplastic neurological syndromes to objectively determine extent and course of disease.

BACKGROUND AND PURPOSE: Paraneoplastic neurological syndromes with Hu-antibodies (Hu-PNS) are immune-mediated disorders in patients with malignancies, most frequently small cell lung cancer, affecting both the peripheral and central nervous system (CNS). In Hu-PNS, brainstem and cerebellar involvement are common. Here, we assessed whether eye-movement disturbances can be used for diagnosis and monitoring of CNS involvement in Hu-PNS. METHODS: Twenty-nine patients with Hu-PNS (17 females; mean age, 63.2 years,) and 14 healthy age-matched controls (seven females; mean age, 60.2 years) were included. Saccadic and smooth pursuit eye movements in response to visual stimuli were recorded with video-oculography. Eye movements were scored quantitatively (number of correction saccades, saccadic intrusions, and saccades during fixation period) and qualitatively by two eye-movement experts. In 20 patients, up to three follow-up measurements were made during subsequent hospital visits with fixed 4-week intervals. Disease course was assessed using the modified Rankin Scale. RESULTS: Eye movements were disturbed in 26 of 29 Hu-PNS patients, with horizontal eye movements being in general more impaired. Moreover, in 12 of the 14 Hu-PNS patients without clinical CNS involvement, eye movements were disturbed. Changes in eye-movement control over a period of up to 12 weeks were significantly correlated with the clinical response to treatment (ρ = 0.52, p = 0.02). CONCLUSIONS: Hu-PNS often affects eye-movement control, also in the absence of CNS signs or symptoms. Eye-movement recordings in Hu-PNS patients might be a useful tool to objectively monitor progression and treatment efficacy in Hu-PNS patients.