mRNA Processing Events Research Articles

RNF20-mediated transcriptional pausing and VEGFA splicing orchestrate vessel growth

Signal-responsive gene expression is essential for vascular development, yet the mechanisms integrating signaling inputs with transcriptional activities are largely unknown. Here we show that RNF20, the primary E3 ubiquitin ligase for histone H2B, plays a multifaceted role in sprouting angiogenesis. RNF20 mediates RNA polymerase (Pol II) promoter-proximal pausing at genes highly paused in endothelial cells, involved in VEGFA signaling, stress response, cell cycle control and mRNA splicing. It also orchestrates large-scale mRNA processing events that alter the bioavailability and function of critical pro-angiogenic factors, such as VEGFA. Mechanistically, RNF20 restricts ERG-dependent Pol II pause release at highly paused genes while binding to Notch1 to promote H2B monoubiquitination at Notch target genes and Notch-dependent gene expression. This balance is crucial, as loss of Rnf20 leads to uncontrolled tip cell specification. Our findings highlight the pivotal role of RNF20 in regulating VEGF–Notch signaling circuits during vessel growth, underscoring its potential for therapeutic modulation of angiogenesis.

The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis.

Processing bodies (P-bodies) are cytoplasmic membrane-less organelles which host multiple mRNA processing events. While the fundamental principles of P-body organization are beginning to be elucidated in vitro, a nuanced understanding of how their assembly is regulated in vivo remains elusive. Here, we investigate the potential link between ER exit sites and P-bodies in Drosophila melanogaster egg chambers. Employing a combination of live and super-resolution imaging, we found that P-bodies associated with ER exit sites are larger and less mobile than cytoplasmic P-bodies, indicating that they constitute a distinct class of P-bodies which are more mature than their cytoplasmic counterparts. Moreover, we demonstrate that altering the composition of ER exit sites has differential effects on core P-body proteins (Me31B, Cup, and Trailer Hitch) suggesting a potential role for ER exit sites in P-body organization. We further show that in the absence of ER exit sites, P-body integrity is compromised and the stability and translational repression efficiency of the maternal mRNA, oskar, are reduced. Finally, we show that ER stress is communicated to P-bodies via ER exit sites, highlighting the pivotal role of ER exit sites as a bridge between membrane-bound and membrane-less organelles in ER stress response. Together, our data unveils the significance of ER exit sites not only in governing P-body organization, but also in facilitating inter-organellar communication during stress, potentially bearing implications for a variety of disease pathologies.

MRNA isoform balance in neuronal development and disease.

Balanced mRNA isoform diversity and abundance are spatially and temporally regulated throughout cellular differentiation. The proportion of expressed isoforms contributes to cell type specification and determines key properties of the differentiated cells. Neurons are unique cell types with intricate developmental programs, characteristic cellular morphologies, and electrophysiological potential. Neuron-specific gene expression programs establish these distinctive cellular characteristics and drive diversity among neuronal subtypes. Genes with neuron-specific alternative processing are enriched in key neuronal functions, including synaptic proteins, adhesion molecules, and scaffold proteins. Despite the similarity of neuronal gene expression programs, each neuronal subclass can be distinguished by unique alternative mRNA processing events. Alternative processing of developmentally important transcripts alters coding and regulatory information, including interaction domains, transcript stability, subcellular localization, and targeting by RNA binding proteins. Fine-tuning of mRNA processing is essential for neuronal activity and maintenance. Thus, the focus of neuronal RNA biology research is to dissect the transcriptomic mechanisms that underlie neuronal homeostasis, and consequently, predispose neuronal subtypes to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

At the Intersection of Major and Minor Spliceosomes: Crosstalk Mechanisms and Their Impact on Gene Expression.

Many eukaryotic species contain two separate molecular machineries for removing non-coding intron sequences from pre-mRNA molecules. The majority of introns (more than 99.5% in humans) are recognized and excised by the major spliceosome, which utilizes relatively poorly conserved sequence elements at the 5′ and 3′ ends of the intron that are used for intron recognition and in subsequent catalysis. In contrast, the minor spliceosome targets a rare group of introns (approximately 0.5% in humans) with highly conserved sequences at the 5′ and 3′ ends of the intron. Minor introns coexist in the same genes with major introns and while the two intron types are spliced by separate spliceosomes, the two splicing machineries can interact with one another to shape mRNA processing events in genes containing minor introns. Here, we review known cooperative and competitive interactions between the two spliceosomes and discuss the mechanistic basis of the spliceosome crosstalk, its regulatory significance, and impact on spliceosome diseases.

A cell culture model/mimic to investigate the biochemical consequences of cdkal1 mutations that result in unprocessed, non‐functional insulin and Type 2 Diabetes Mellitus

The primary objective of the project is to develop a cell culture model that mimics the pancreatic beta islets to study the biochemical consequences of single nucleotide polymorphisms (SNPs) found in the intron-5 of cdkal1 gene which codes for a tRNA modification enzyme. SNPs in cdkal1 gene has been pointed out for its role in the development of Type 2 Diabetes Mellitus (T2DM) from past few years by human Genome Wide Association Studies. Still we lack a model system to investigate the consequences of cdkal1 mutations that result in unprocessed, non-functional insulin leading to the development of T2DM. Hypothesis Incorrectly processed cdkal1 mRNA results in untranslated or incorrectly translated Cdkal1 protein. Cdkal1 protein is a thiomethyl transferase that adds an ms2 moiety to the already modified N6-threonyl carbamoyl adenosine (t6A37) in the 37 position of tRNA. Improperly processed Cdkal1 protein will not be able to modify tRNALys3. Proinsulin will not be correctly translated and processed resulting in little or no insulin secreted from the β- cells in pancreatic islets. To uncover the pathogenesis of the human cdkal1-SNP associated T2DM, the current project will produce a cell culture model of the cdkal1 gene SNP-associated human T2DM. With the model, we will be able to discover the biochemical properties of the insulin, if translated at all. In a cdkal1-knockout (KO) cells, we will rescue the insulin production with WT human cdkal1, and then discover the effects of intron-5 SNPs of the cdkal1 gene. SNPs could affect mRNA processing. Methods and preliminary results We have successfully knocked down the cdkal1 gene in a murine β-cell line (NIT1) inducible for insulin. Our recent knockdown studies in NIT1 cell lines showed that under KCl insulin induction, unprocessed insulin mRNA increases by ~40% relative to mature insulin mRNA (Fig. B) and cdkal1 mRNA is reduced by ~40%. Subsequent reduction in tRNA ms2 modification and insulin production was also observed upon cdkal1 knockdown. As 100% gene knockout is required to study the effects of SNPs, we now have a murine β-cell line (MIN-cdkal1-null); the parent of that cell line (MIN-cdkal1-het) produces insulin assessed with a glucose- stimulated insulin release (GSIR) assay. The MIN-cdkal1-null having a cdkal1 knockout will be an advantageous tool to evaluate the state of insulin production when Cdkal1 is not modifying tRNALys3. A variant of mini-gene reporter constructs carrying potential SNPs have been designed and will be expressed and analyzed in knockout cell lines to evaluate cdkal1 mRNA processing and splicing events. We are positioned to answer important questions that connect Cdkal1 protein biochemistry to T2DM like, are the human intronic SNPs of the cdkal1 gene cause incorrect processing of cdkal1 mRNA? What is the status of the insulin produced, if at all translated - is proinsulin unprocessed, aggregated or degraded and not secreted? In the long-term, the results of this study will stimulate new insights into the human health implications of tRNA modification enzymes and the role(s) of modifications not just in the pathogenesis of cdkal1 SNP-associated T2DM, but also in a variety of human diseases.

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.

Mitochondrial mRNA Processing in the Chlorophyte Alga Pediastrum duplex and Streptophyte Alga Chara vulgaris Reveals an Evolutionary Branch in Mitochondrial mRNA Processing

Mitochondria carry the remnant of an ancestral bacterial chromosome and express those genes with a system separate and distinct from the nucleus. Mitochondrial genes are transcribed as poly-cistronic primary transcripts which are post-transcriptionally processed to create individual translationally competent mRNAs. Algae post-transcriptional processing has only been explored in Chlamydomonas reinhardtii (Class: Chlorophyceae) and the mature mRNAs are different than higher plants, having no 5′ UnTranslated Regions (UTRs), much shorter and more variable 3′ UTRs and polycytidylated mature mRNAs. In this study, we analyzed transcript termini using circular RT-PCR and PacBio Iso-Seq to survey the 3′ and 5′ UTRs and termini for two green algae, Pediastrum duplex (Class: Chlorophyceae) and Chara vulgaris (Class: Charophyceae). This enabled the comparison of processing in the chlorophyte and charophyte clades of green algae to determine if the differences in mitochondrial mRNA processing pre-date the invasion of land by embryophytes. We report that the 5′ mRNA termini and non-template 3′ termini additions in P. duplex resemble those of C. reinhardtii, suggesting a conservation of mRNA processing among the chlorophyceae. We also report that C. vulgaris mRNA UTRs are much longer than chlorophytic examples, lack polycytidylation, and are polyadenylated similar to embryophytes. This demonstrates that some mitochondrial mRNA processing events diverged with the split between chlorophytic and streptophytic algae.

URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway

The TREX complex integrates information from nuclear mRNA processing events to ensure the timely export of mRNA to the cytoplasm. In humans, UAP56 and its paralog URH49 form distinct complexes, the TREX complex and the AREX complex, respectively, which cooperatively regulate the expression of a specific set of mRNA species on a genome wide scale. The difference in the complex formation between UAP56 and URH49 are thought to play a critical role in the regulation of target mRNAs. To date, the underlying mechanism remains poorly understood. Here we characterize the formation of the TREX complex and the AREX complex. In the ATP depleted condition, UAP56 formed an Apo-TREX complex containing the THO subcomplex but not ALYREF and CIP29. URH49 formed an Apo-AREX complex containing CIP29 but not ALYREF and the THO subcomplex. However, with the addition of ATP, both the Apo-TREX complex and the Apo-AREX complex were remodeled to highly similar ATP-TREX complex containing the THO subcomplex, ALYREF and CIP29. The knockdown of URH49 caused a reduction in its target mRNAs and a cytokinesis failure. Similarly, cytokinesis abnormality was observed in CIP29 knockdown cells, suggesting that CIP29 belongs to the URH49 regulated mRNA export pathway. Lastly, we confirmed that the export of mRNA in URH49-dependent pathway is achieved by NXF1, which is also observed in UAP56-dependent pathway. Our studies propose an mRNA export model that the mRNA selectivity depends on the Apo-form TREX/AREX complex, which is remodeled to the highly similar ATP-form complex upon ATP loading, and integrated to NXF1.

Alternative mRNA splicing in cancer immunotherapy.

Immunotherapies are yielding effective treatments for several previously untreatable cancers. Still, the identification of suitable antigens specific to the tumour that can be targets for cancer vaccines and T cell therapies is a challenge. Alternative processing of mRNA, a phenomenon that has been shown to alter the proteomic diversity of many cancers, may offer the potential of a broadened target space. Here, we discuss the promise of analysing mRNA processing events in cancer cells, with an emphasis on mRNA splicing, for the identification of potential new targets for cancer immunotherapy. Further, we highlight the challenges that must be overcome for this new avenue to have clinical applicability.

Networks of mRNA Processing and Alternative Splicing Regulation in Health and Disease.

mRNA processing events introduce an intricate layer of complexity into gene expression processes, supporting a tremendous level of diversification of the genome's coding and regulatory potential, particularly in vertebrate species. The recent development of massive parallel sequencing methods and their adaptation to the identification and quantification of different RNA species and the dynamics of mRNA metabolism and processing has generated an unprecedented view over the regulatory networks that are established at this level, which contribute to sustain developmental, tissue specific or disease specific gene expression programs. In this chapter, we provide an overview of the recent evolution of transcriptome profiling methods and the surprising insights that have emerged in recent years regarding distinct mRNA processingevents - from the 5' end to the 3' end of the molecule.

Small-Molecule Targeting of Musashi RNA-Binding Activity in Acute Myeloid Leukemia

RNA-binding proteins (RBPs) play critical roles in cell homeostasis by controlling gene expression post-transcriptionally, contributing to mRNA processing events (splicing, polyadenylation, localization, stability, export and translation). The involvement of RBPs to tumorigenesis, through genetic perturbation or epigenetic dysregulation, has been found in a variety of human cancers. The RBP MUSASHI-2 (MSI2) contributes to the pathogenesis of a spectrum of solid tumors and hematologic malignancies and predicts a worse clinical outcome in patients with myeloid and acute lymphoblastic leukemia (MDS, AML and ALL). Thus, MSI2 has been proposed as a putative biomarker for diagnosis as well as a potential therapeutic target for AML. However, there are currently no specific inhibitors for MSI.Previous work from our lab reported a Fluorescent Polarization (FP) screen with 6,208 compounds identifying small-molecules with MSI RNA-binding inhibition activity. Here, we characterize Ro 08-2750 (Ro), best FP screen hit, as a MSI RNA-competitive inhibitor. Electrophoresis Mobility Shift Assays (EMSA) demonstrated Ro inhibition of MSI2-RNA complexes formation. MicroScale Thermophoresis (MST) interaction studies showed that the compound interacts with MSI2 full-length and RNA-Recognition Motif 1 (RRM1) with μM affinity and with nearly 20-fold lower KD to an RBP control (SYNCRIP). We obtained the crystal structure of MSI2 RRM1 at 1.7Å and docking and mutagenesis validation confirmed K22, F66, F97 and R100 as crucial binding residues in the RNA-binding pocket. To further prove structure activity relationship, we used two chemical analogs: Ro-OH, an alcohol derivative of the Ro's aldehyde, showed 10-fold reduced activity and Ro-NGF, containing the Ro isoalloxazine scaffold, showed no binding or activity in vitro. Of note, in proliferation assays Ro EC50 was 2.6±0.1 μM in MLL-AF9 bone marrow cells and an average of 8.4±1.1 μM in MOLM13 and K562 human AML cells, whereas RoOH and RoNGF showed 10-fold or >50 μM EC50, respectively. Ro significantly reduced binding of MSI2 to its mRNA targets (such as cMYC, CDKN1A or SMAD3) in an RNA-IP and a direct effect in their protein translation in human leukemia cells. RNA-sequencing of 4h Ro treated MOLM13 and K562 AML cells resulted in gene expression changes that enriched for the gene expression profiling after shRNA mediated depletion of MSI2 in CML-BC and AML cell lines. Ro demonstrated a significant therapeutic index abolishing MLL-AF9+ BM colony formation at concentrations that did not affect the plating efficiency of normal Lin-Sca+cKit+ (LSK) cells. Similarly, Ro demonstrated differential sensitivity in three AML patient samples colony formation compared to normal human CD34+ cord blood cells. Finally, we sought to determine Ro in vivo activity by using an aggressive murine MLL-AF9 murine leukemia model. Acute treatment (4h and 12hr) with 13.75 mg/kg Ro in DMSO reduced c-KIT protein abundance and intracellular c-MYC. Administration of the same Ro dose every 3 days was well tolerated and showed a significant reduction in spleen weights, white blood cell counts and c-MYC levels compared to the controls. These data provide the feasibility that targeting MSI in vivo could have therapeutic efficacy in AML.This study identifies and characterizes Ro 08-2750 as a compound selectively inhibiting the oncogenic RNA-binding activity of MSI in myeloid leukemia. Ro targeting an RRM motif to block RNA activity represents a valuable proof of concept for the general inhibition of these class of RNA regulators. Overall, we provide a framework to identify and test novel RBP inhibitors thus validating this class of proteins as chemically “druggable” novel therapeutic targets in cancer. DisclosuresChodera:Schrödinger: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing

BackgroundThe multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing.ResultsIn MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells.ConclusionsOur findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

Splicing regulation by long noncoding RNAs.

Massive high-throughput sequencing techniques allowed the identification of thousands of noncoding RNAs (ncRNAs) and a plethora of different mRNA processing events occurring in higher organisms. Long ncRNAs can act directly as long transcripts or can be processed into active small si/miRNAs. They can modulate mRNA cleavage, translational repression or the epigenetic landscape of their target genes. Recently, certain long ncRNAs have been shown to play a crucial role in the regulation of alternative splicing in response to several stimuli or during disease. In this review, we focus on recent discoveries linking gene regulation by alternative splicing and its modulation by long and small ncRNAs.

N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing.

N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3΄ end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.

Cbp80 is needed for the expression of piRNA components and piRNAs.

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.

TDP-43 and FUS en route from the nucleus to the cytoplasm.

Misfolded or mislocalized RNA-binding proteins (RBPs) and, consequently, altered mRNA processing, can cause neuronal dysfunction, eventually leading to neurodegeneration. Two prominent examples are the RBPs TAR DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS), which form pathological messenger ribonucleoprotein aggregates in patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating neurodegenerative disorders. Here, we review the multiple functions of TDP-43 and FUS in mRNA processing, both in the nucleus and in the cytoplasm. We discuss how TDP-43 and FUS may exit the nucleus and how defects in both nuclear and cytosolic mRNA processing events, and possibly nuclear export defects, may contribute to neurodegeneration and ALS/FTD pathogenesis.

The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II.

RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription.

Identification of learning-induced changes in protein networks in the hippocampi of a mouse model of Alzheimer's disease.

Memory loss is the most profound clinical manifestation in Alzheimer's disease (AD); however, the molecular mechanisms underlying these deficits are poorly understood. Identification of the molecular pathways involved in the onset of cognitive deficits may lead to the identification of key events in the pathogenesis of AD. Using isobaric tags for relative and absolute quantitation (iTRAQ) and proteomic methods, here we identified learning-induced changes in the hippocampal proteome of non-transgenic (NonTg) and 3 × Tg-AD mice, a widely used animal model of AD. We found that expression of 192 proteins was differentially regulated by learning in NonTg mice. Notably, of these 192 proteins, only 28 were also differentially regulated by learning in 3 × Tg-AD mice, whereas the levels of 164 proteins were uniquely changed in NonTg mice but not in 3 × Tg-AD mice. These data suggest that during learning, 3 × Tg-AD mice fail to differentially regulate 164 proteins. Gene ontology and protein interaction analyses indicated that these proteins were overrepresented in RNA processing, specifically RNA transport, splicing and mRNA translation initiation pathways. These findings suggest that mRNA-processing events that take place during learning and memory are significantly altered in 3 × Tg-AD mice.

Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes.

RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV–Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.

HnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation.

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5' untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1-mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development.