Regulatory RNA-binding Proteins Research Articles

Selective inhibition of miR-92 in hippocampal neurons alters contextual fear memory.

Post-transcriptional gene regulation mediated by microRNAs (miRNAs) is implicated in memory formation; however, the function of miR-92 in this regulation is uncharacterized. The present study shows that training mice in contextual fear conditioning produces a transient increase in miR-92 levels in the hippocampus and decreases several miR-92 gene targets, including: (i) the neuronal Cl(-) extruding K(+) Cl(-) co-transporter 2 (KCC2) protein; (ii) the cytoplasmic polyadenylation protein (CPEB3), an RNA-binding protein regulator of protein synthesis in neurons; and (iii) the transcription factor myocyte enhancer factor 2D (MEF2D), one of the MEF2 genes which negatively regulates memory-induced structural plasticity. Selective inhibition of endogenous miR-92 in CA1 hippocampal neurons, by a sponge lentiviral vector expressing multiple sequences imperfectly complementary to mature miR-92 under the control of the neuronal specific synapsin promoter, leads to up-regulation of KCC2, CPEB3 and MEF2D, impairs contextual fear conditioning, and prevents a memory-induced increase in the spine density. Taken together, the results indicate that neuronal-expressed miR-92 is an endogenous fine regulator of contextual fear memory in mice.

Inhibition of Caspases Protects Mice from Radiation-induced Oral Mucositis and Abolishes the Cleavage of RNA-binding Protein HuR

The oral mucosal epithelium is typically insulted during chemotherapy and ionizing radiation (IR) therapy and disposed to mucositis, which creates painful inflammation and ulceration in the oral cavity. Oral mucositis alters gene expression patterns, inhibits cellular growth, and initiates cell death in the oral epithelial compartments. Such alterations are governed by several different factors, including transcription factors, RNA-binding proteins, and microRNAs. IR-induced post-transcriptional regulation of RNA-binding proteins exists but is poorly studied in clinically relevant settings. We herein report that the RNA-binding protein human antigen R (HuR) undergoes cleavage modification by caspase-3 following IR-induced oral mucositis and subsequently promotes the expression of the pro-apoptotic factor BAX (Bcl-2-associated X protein), as well as cell death. Further analyses revealed that the HuR cleavage product-1 (HuR-CP1) directly associates and stabilizes the BAX mRNA and concurrently activates the apoptotic pathway. On the other hand, a noncleavable isoform of HuR promotes the clonogenic capacity of primary oral keratinocytes and decreases the effect of IR-induced cell death. Additionally, specific inhibition of caspase-3 by a compound, NSC321205, increases the clonogenic capacity of primary oral keratinocytes and causes increased basal layer cellularity, thickened mucosa, and elevated epithelial cell growth in the tongues of mice with oral mucositis. This protective effect of NSC321205 is mediated by a decrease in caspase-3 activity and the consequent inhibition of HuR cleavage, which reduces the expression of BAX in mice with IR-induced oral mucositis. Thus, we have identified a new molecular mechanism of HuR in the regulation of mRNA turnover and apoptosis in oral mucositis, and our data suggest that blocking the cleavage of HuR enhances cellular growth in the oral epithelial compartment.

RNA-binding proteins in regulation of alternative cleavage and polyadenylation.

Almost all eukaryotic pre-mRNAs are processed at the 3' end by the cleavage and polyadenylation (C/P) reaction, which preludes termination of transcription and gives rise to the poly(A) tail of mature mRNA. Genomic studies in recent years have indicated that most eukaryotic mRNA genes have multiple cleavage and polyadenylation sites (pAs), leading to alternative cleavage and polyadenylation (APA) products. APA isoforms generally differ in their 3' untranslated regions (3' UTRs), but can also have different coding sequences (CDSs). APA expands the repertoire of transcripts expressed from the genome, and is highly regulated under various physiological and pathological conditions. Growing lines of evidence have shown that RNA-binding proteins (RBPs) play important roles in regulation of APA. Some RBPs are part of the machinery for C/P; others influence pA choice through binding to adjacent regions. In this chapter, we review cis elements and trans factors involved in C/P, the significance of APA, and increasingly elucidated roles of RBPs in APA regulation. We also discuss analysis of APA using transcriptome-wide techniques as well as molecular biology approaches.

Zfp36 expression delineates both myeloid cells and cells localized to the fusing neural folds in Xenopus tropicalis.

Regulatory RNA binding proteins allow for specific control of gene expression in a very dynamic manner. In mammals ZFP36, formerly known as Tristetraprolin, controls the inflammatory response by binding to an AU-rich element located in the 3' untranslated region of its target mRNAs. The developping embryo relies on a population of primitive macrophages to ensure proper immunity. Although the role of zfp36 in adult immunity has been extensively studied, its expression in the developing immune system has been poorly documented. Here, we have used whole mount in situ hybridization with a 3' UTR specific probe to address the expression of zfp36 in developing Xenopus tropicalis embryos. We have shown that zfp36 is expressed in two distinct cellular populations. First, it is a new marker of primititive myeloid cells, being coexpressed with the myeloid marker mpo. Therefore this early expression may suggest a role for zfp36 in macrophage differentiation and activation. In addition, a second cell population was found to transiently express zfp36, but not mpo, along the fusing neural folds and may correspond to cells undergoing autophagy during neural tube closure.

NF90 in Posttranscriptional Gene Regulation and MicroRNA Biogenesis

Gene expression patterns are effectively regulated by turnover and translation regulatory (TTR) RNA-binding proteins (RBPs). The TTR-RBPs control gene expression at posttranscriptional levels, such as pre-mRNA splicing, mRNA cytoplasmic export, turnover, storage, and translation. Double-stranded RNA binding proteins (DSRBPs) are known to regulate many processes of cellular metabolism, including transcriptional control, translational control, mRNA processing and localization. Nuclear factor 90 (NF90), one of the DSRBPs, is abundantly expressed in vertebrate tissue and participates in many aspects of RNA metabolism. NF90 was originally purified as a component of a DNA binding complex which binds to the antigen recognition response element 2 in the interleukin 2 promoter. Recent studies have provided us with interesting insights into its possible physiological roles in RNA metabolism, including transcription, degradation, and translation. In addition, it was shown that NF90 regulates microRNA expression. In this review, we try to focus on the function of NF90 in posttranscriptional gene regulation and microRNA biogenesis.

Altered Regulation of ELAVL1/HuR in HLA-B27–Expressing U937 Monocytic Cells

ObjectiveTo investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response.MethodsU937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA–B27, or mutated HLA–B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA.ResultsFull length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27.ConclusionResults show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

Prediction of regulatory factor X1 binding sites in promoters of RNA-binding proteins genes in mouse brain.

There is an increasing interest in the role of regulatory factor X1 (RFX1) and RNA-binding proteins (RBPs) during neural development. However, there are few reports about their interaction. We extracted RNA and performed reverse transcription polymerase chain reaction (RT-PCR) to identify RFX1 expression in imprinting control region (ICR) mouse tissues, analyzed RFX1 domains and motif consensus by comparing public databases on the Internet, tested the motif consensus with affinity-capture and western blotting experiments with mouse brain tissue, and predicted the binding sites of RFX1 in promoter regions of mouse RBPs genes. The expression of RFX1 was higher in embryonic brain compared to embryonic kidney, heart, and liver, and its expression level was relatively stable and higher in mouse embryonic brain than neonatal brain. RFX1 had several domains, including domain A as an activation domain, DBD as a DNA binding domain, domain B and C which played an important role in dimerization, and domain D as dimerization domain. RFX1 had three different profiles motif consensus RFX1M00281, RFX1M00280, and RFX1 (EF-C) M00626. There were 79 RFX1 binding sites at the promoters of 65 of 323 RBPs genes. RFX1 as regulatory factor will have putative important regulating role in the expression of RBPs genes during embryonic development of mouse brain.

DNA-Damage-Induced Nuclear Export of Precursor MicroRNAs Is Regulated by the ATM-AKT Pathway

Expression of microRNAs (miRNAs) involves transcription of miRNA genes and maturation of the primary transcripts. Recent studies have shown that posttranscriptional processing of primary and precursor miRNAs is induced after DNA damage through regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP. However, little is known about the regulation of nuclear export of pre-miRNAs in the DNA-damage response, a critical step in miRNA maturation. Here, we show that nuclear export of pre-miRNAs is accelerated after DNA damage in an ATM-dependent manner. The ATM-activated AKT kinase phosphorylates Nup153, a key component of the nucleopore, leading to enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings define an important role of DNA-damage signaling in miRNA transport and maturation.

Identification of regulatory RNA binding proteins that mediate alternative polyadenylation in cardiac myocytes

IntroductionCardiovascular disease is the leading cause of death in the US with coronary heart disease (ischemia/reperfusion; I/R injury) accounting for approximately half of these deaths. Recent data from our lab suggests that alternative polyadenylation (APA) plays a role in cardiac gene expression following I/R via modulation of mRNA 3′UTR length, leading to the inclusion or exclusion of regulatory sequence elements. The goal of this work is to identify specific RNA binding proteins (RBPs) that are likely to mediate APA in the myocardium.MethodsThe 3′UTR of the heat shock protein 70.3 (Hsp70.3) mRNA was randomly biotinylated throughout and incubated with cardiac protein extract associated with differential APA of the Hsp70.3 3′UTR. Biotinylated RNA and RBPs were co‐precipitated and bound RBPs were identified using mass spectrometry.ResultsWe identified a total of 45 known RBPs interacting with the Hsp70.3 3′UTR. Of these, 9 unique RBPs were found to bind only under conditions associated with a long 3′UTR, whereas 10 unique RBPs were found to bind only in association with APA truncation of the 3′UTR. Our results identified RBPs previously indicated to play a role in APA and/or mRNA degradation as well as RBPs whose function remains unknown. Work is ongoing to determine the functional role of these RBPs in alternative polyadenylation and cardiac I/R injury.This work was partially funded by a UC CCTST Grant (MT).

TDP-43 high throughput screening analyses in neurodegeneration: Advantages and pitfalls

Dysfunctions in RNA processing and in particular the aberrant regulation of RNA binding proteins (RBPs) have recently been shown to play a fundamental role in the pathogenesis of neurodegenerative diseases. Understanding the pathogenic mechanisms involved will require the elucidation of the role(s) played by these RBPs in the general cell metabolism and neuronal survival in particular. In the past, the preferred approach has been to determine first of all the functional properties of the factor(s) of interest and then use this knowledge to determine targets in biologically relevant events. More recently, novel experimental approaches such as microarrays, RNA-seq and CLIP-seq have also become very popular to study RBPs. The advantage of these approaches, collectively known as high throughput screening (HTS), is their ability to determine gene expression changes or RNA/protein targets at a global cellular level. In theory, HTS strategies should be ideal for uncovering novel functional roles/targets of any RBP inside the cell. In practice, however, there are still difficulties in getting a coherent picture from all the huge amount of data they generate, frequently not validated experimentally and thus of unknown value. They may even act unfavorably towards a specific increase of knowledge of RBP functions, as the incomplete results are taken as solid data. In this work we will illustrate as an example the use of the HTS methodologies to characterize the interactions of a specific RBP: TDP-43. The multiple functions of this protein in RNA processing and its involvement in the pathogenesis of several forms of amyotrophic lateral sclerosis, frontotemporal lobar degeneration and other neurodegenerative diseases make it an excellent substrate for our analysis of the various advantages and limitations of different HTS experimental approaches.

A Compendium of Caenorhabditis elegans RNA Binding Proteins Predicts Extensive Regulation at Multiple Levels

Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs.

RNA Binding Protein/RNA Element Interactions and the Control of Translation

A growing body of work demonstrates the importance of post-transcriptional control, in particular translation initiation, in the overall regulation of gene expression. Here we focus on the contribution of regulatory elements within the 5’ and 3’ untranslated regions of mRNA to gene expression in eukaryotic cells including terminal oligopyrimidine tracts, internal ribosome entry segments, upstream open reading frames and cytoplasmic polyadenylation elements. These mRNA regulatory elements may adopt complex secondary structures and/or contain sequence motifs that allow their interaction with a variety of regulatory proteins, RNAs and RNA binding proteins, particularly hnRNPs. The resulting interactions are context-sensitive, and provide cells with a sensitive and fast response to cellular signals such as hormone exposure or cytotoxic stress. Importantly, an increasing number of diseases have been identified, particularly cancers and those associated with neurodegeneration, which originate either from mutation of these regulatory motifs, or from deregulation of their cognate binding partners.

When Ribonucleases Come into Play in Pathogens: A Survey of Gram-Positive Bacteria

It is widely acknowledged that RNA stability plays critical roles in bacterial adaptation and survival in different environments like those encountered when bacteria infect a host. Bacterial ribonucleases acting alone or in concert with regulatory RNAs or RNA binding proteins are the mediators of the regulatory outcome on RNA stability. We will give a current update of what is known about ribonucleases in the model Gram-positive organism Bacillus subtilis and will describe their established roles in virulence in several Gram-positive pathogenic bacteria that are imposing major health concerns worldwide. Implications on bacterial evolution through stabilization/transfer of genetic material (phage or plasmid DNA) as a result of ribonucleases' functions will be covered. The role of ribonucleases in emergence of antibiotic resistance and new concepts in drug design will additionally be discussed.

12 INVITED MicroRNAs and Regulatory RNA Binding Proteins in Cancer

12 INVITED MicroRNAs and Regulatory RNA Binding Proteins in Cancer

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Role of RNA-Binding Proteins in MAPK Signal Transduction Pathway

Mitogen-activated protein kinases (MAPKs), which are found in all eukaryotes, are signal transducing enzymes playing a central role in diverse biological processes, such as cell proliferation, sexual differentiation, and apoptosis. The MAPK signaling pathway plays a key role in the regulation of gene expression through the phosphorylation of transcription factors. Recent studies have identified several RNA-binding proteins (RBPs) as regulators of MAPK signaling because these RBPs bind to the mRNAs encoding the components of the MAPK pathway and regulate the stability of their transcripts. Moreover, RBPs also serve as targets of MAPKs because MAPK phosphorylate and regulate the ability of RBPs to bind and stabilize target mRNAs, thus controlling various cellular functions. In this review, we present evidence for the significance of the MAPK signaling in the regulation of RBPs and their target mRNAs, which provides additional information about the regulatory mechanism underlying gene expression. We further present evidence for the clinical importance of the posttranscriptional regulation of mRNA stability and its implications for drug discovery.

Proteome-Wide Search Reveals Unexpected RNA-Binding Proteins in Saccharomyces cerevisiae

The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs - most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation.

Brk/PTK6 signaling in normal and cancer cell models

Breast tumor kinase (Brk), also termed PTK6, is known to function in cell-type and context-dependent processes governing normal differentiation. However, in tumors in which Brk is overexpressed, this unusual soluble tyrosine kinase is emerging as a mediator of cancer cell phenotypes, including increased proliferation, survival, and migration. Nuclear and cytoplasmic substrates phosphorylated by Brk include a collection of regulatory RNA-binding proteins, adaptor molecules that link Brk to signaling pathways generally associated with the activation of growth factor receptors, and Signal Transducers and Activators of Transcription (STAT) molecules that are direct regulators of gene expression. Understanding Brk-dependent regulation of these key signaling pathways and how they influence cancer cell behavior is predicted to inform the development of improved 'targeted' cancer therapies and may provide insight into ways to avoid chemo-resistance to established treatments.

Emerging tools for exploring RNA regulation (LL4-3)

RNA regulation is shifting to the forefront of gene expression in part because it has been difficult to study in the past; but new tools are making it more amenable to exploration on several levels from splicing to export to stability and translation. The field of RNA-protein interactions emerged with the cloning of cDNAs expressing RNA-binding proteins (RBPs) while the enzymes to synthesize cRNA and to map binding sites of RBPs with precision became available and provided important information related to RBP functions in splicing and translational control. In the early 1990s it was assumed that gene expression was solely coordinated at transcription, and that RNA-protein interactions were essentially single events. However, by using in vitro selection of naturally occurring sequences, it was demonstrated that RBPs could be multi-targeted on a larger scale to bind similar sequence elements in the 3′ UTRs of hundreds of brain-derived mRNAs. Later, microarrays allowed investigators to assess the steady-state levels of transcripts and were subsequently used to identify functionally related mRNAs associated with RBPs that govern their localization, coordination and dynamics. Many of the tools used in the discovery of coherent groups of mRNAs coordinated by RBPs in the immune system will be described. Moreover, the use of microarrays and next generation high-throughput sequencing procedures for detecting functional interactions of mRNAs and noncoding RNAs with regulatory RBPs on a genome-wide scale during immune responses will be presented.

Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.