RNA-binding Target Research Articles

Structure and Dynamics of the CCCH-Type Tandem Zinc Finger Domain of POS-1 and Implications for RNA Binding Specificity.

CCCH-type tandem zinc finger (TZF) motifs are found in many RNA-binding proteins involved in regulating mRNA stability, translation, and splicing. In Caenorhabditis elegans, several RNA-binding proteins that regulate embryonic development and cell fate determination contain CCCH TZF domains, including POS-1. Previous biochemical studies have shown that despite high levels of sequence conservation, POS-1 recognizes a broader set of RNA sequences compared to the human homologue tristetraprolin. However, the molecular basis of these differences remains unknown. In this study, we refined the consensus RNA sequence and determined the differing binding specificities of the two zinc fingers of POS-1. We also determined the solution structure and characterized the internal dynamics of the TZF domain of POS-1. From the structure, we identified unique features that define the RNA binding specificity of POS-1. We also observed that the TZF domain of POS-1 is in equilibrium between interconverting conformations. Transitions between these conformations require internal motions involving many residues with correlated dynamics in each ZF. We propose that the correlated dynamics are necessary to allow allosteric communication between the nucleotide-binding pockets observed in the N-terminal ZF. Our study shows that both the structure and conformational plasticity of POS-1 are important in ensuring recognition of its RNA binding targets.

Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation

RNA interactome studies have revealed that hundreds of zinc-finger proteins (ZFPs) are candidate RNA-binding proteins (RBPs), yet their RNA substrates and functional significance remain largely uncharacterized. Here, we present a systematic multi-omics analysis of the DNA- and RNA-binding targets and regulatory roles of more than 100 ZFPs representing 37 zinc-finger families. We show that multiple ZFPs are previously unknown regulators of RNA splicing, alternative polyadenylation, stability, or translation. The examined ZFPs show widespread sequence-specific RNA binding and preferentially bind proximal to transcription start sites. Additionally, several ZFPs associate with their targets at both the DNA and RNA levels. We highlight ZNF277, a C2H2 ZFP that binds thousands of RNA targets and acts as a multi-functional RBP. We also show that ZNF473 is a DNA/RNA-associated protein that regulates the expression and splicing of cell cycle genes. Our results reveal diverse roles for ZFPs in transcriptional and post-transcriptional gene regulation.

Fascin actin-bundling protein 1 regulates non-small cell lung cancer progression by influencing the transcription and splicing of tumorigenesis-related genes

Background High mortality rates are prevalent among patients with non-small-cell lung cancer (NSCLC), and effective therapeutic targets are key prognostic factors. Fascin actin-bundling protein 1 (FSCN1) promotes NSCLC; however, its role as an RNA-binding protein in NSCLC remains unexplored. Therefore, we aimed to explore FSCN1 expression and function in A549 cells. Method We screened for alternative-splicing events and differentially expressed genes (DEGs) after FSCN1 silence via RNA-sequencing (RNA-seq). FSCN1 immunoprecipitation followed by RNA-seq were used to identify target genes whose mRNA expression and pre-mRNA alternative-splicing levels might be influenced by FSCN1. Results Silencing FSCN1 in A549 cells affected malignant phenotypes; it inhibited proliferation, migration, and invasion, and promoted apoptosis. RNA-seq analysis revealed 2,851 DEGs and 3,057 alternatively spliced genes. Gene ontology-based functional enrichment analysis showed that downregulated DEGs and alternatively splicing genes were enriched for the cell-cycle. FSCN1 promoted the alternative splicing of cell-cycle-related mRNAs involved in tumorigenesis (i.e., BCCIP, DLGAP5, PRC1, RECQL5, WTAP, and SGO1). Combined analysis of FSCN1 RNA-binding targets and RNA-seq data suggested that FSCN1 might affect ACTG1, KRT7, and PDE3A expression by modulating the pre-mRNA alternative-splicing levels of NME4, NCOR2, and EEF1D, that were bound to long non-coding RNA transcripts (RNASNHG20, NEAT1, NSD2, and FTH1), which were highly abundant. Overall, extensive transcriptome analysis of gene alternative splicing and expression levels was performed in cells transfected with FSCN1 short-interfering RNA. Our data provide global insights into the regulatory mechanisms associated with the roles of FSCN1 and its target genes in lung cancer.

The Identification of NIFK’s RNA Binding Targets and Its Prognostic Values in Lung Cancer

BackgroundWe previously identified nucleolar protein interacting with the FHA domain of pKi‐67 (NIFK) as a poor prognostic marker in lung cancer. Furthermore, NIFK overexpression promotes cancer metastasis via decreasing casein kinase 1α (CK1α) expression, a suppressor of pro‐metastatic TCF4/β‐catenin signaling. We further observe the axis is induced by the downregulation of a novel transcription factor RUNX1. It is also known for NIFK’s biological function in regulating RNA metabolism via RNA recognition motif (RRM). However, a comprehensive analysis for identifying the RNA binding targets has not yet been reported. In this study, we aim to unravel the interactive mechanism and to characterize its potential clinical value in cancer patient cohort.MethodNIFK is pointed out to possess RNA binding function via RRM. RNA immunoprecipitation assay was performed using specific NIFK antibody in lung cancer PC13 and A549 cells after NIFK overexpression. RNA targets were analyzed by RT‐PCR and Next Generation Sequencing (NGS). In addition, expression levels were investigated by IHC staining and omics data mining. The association of the respective expression with patient survival outcome were explored in lung cancer cohorts.ResultsWe observe the increase in amount of RNA binding targets after NIFK overexpression respectively in PC13 and A549 cells as comparing with the control. Moreover, NIFK appears to bind with RUNX1 RNA but not CK1α suggesting it is the initial step of NIFK‐mediated TCF4/β‐catenin signaling regulation. NIFK also leads to RUNX1 RNA instability of which mechanism remains to be explored. RUNX1 level is observed to associate with good prognosis in lung cancer datasets (jacob‐00182‐CANDF and GSE13213). Furthermore, direct interactive RNA targets of NIFK are identified by RNA immunoprecipitation and RNA‐Seq, which indicate SYNE2 as one of targets with prognostic significance in lung cancer. Increased SYNE2 is detected upon NIFK overexpression, and the lung cancer patients stratified in high SYNE2 group appear to associate with inferior clinical outcomes.ConclusionWe first show the interactive mechanism of pro‐metastatic molecule NIFK in regulating TCF4/β‐catenin signaling and SYNE2, which might provide insights into therapeutic development for lung cancer patients.

Abstract P028: Proteomic approaches define rocaglates as translation remodelers with multiple protein targets

Abstract Intro: Deregulated protein synthesis is a common trait across solid and hematologic malignancies and an attractive target for cancer therapy. Rocaglates compounds that inhibit eukaryotic initiation factor 4A1 (eIF4A1), the essential DEAD-box RNA helicase that resolves mRNA 5’UTR secondary structures during cap-dependent translation initiation. Rocaglates’ unique mechanism of action causes sequence-selective mRNA binding by eIF4A1, clamping the inactive helicase onto the transcript. This suppresses translation globally and affects many oncogenic and pro-survival transcripts in particular. Zotatifin, the first-in class synthetic rocaglate, is currently in Phase I clinical trials for the treatment of solid tumors and as an antiviral against SARS-CoV2. Currently, eIF4A1 and DDX3 are the only reported targets of rocaglate-mediated RNA clamping. Employing unbiased proteomic approaches, we have discovered that rocaglates, thought to act as pure eIF4A/translation inhibitors, extensively remodel the translation machinery and translatome. Additionally, mass-spec interrogation for proteins interacting with specific RNA sequences reveals novel targets of rocaglate-mediated, sequence-specific RNA clamping. Methods: We conducted original mass-spectrometry analyses of translational reprogramming by rocaglates. TMT-pSILAC assessed acute changes in protein production, while MATRIX, which captures high-resolution profiles of the translation machinery, revealed translation factors that drive reprogramming in response to rocaglate exposure. We validated results biochemically, in cellulo, and in vivo using patient-derived xenograft (PDX) mouse models. To probe existing and novel rocaglate RNA-clamping targets, we developed unbiased “clampome” assays – in cellulo protein-RNA-pull downs followed by mass-spec analysis of proteins with increased binding to RNA in the presence of rocaglates. Results: We find rocaglates, including zotatifin, have effects far more complex than simple “translational inhibition” as currently defined. Indeed, translatome analysis by TMT-pSILAC revealed myriad up-regulated proteins that drive hitherto unrecognized cytotoxic mechanisms. The GEF-H1 guanine exchange factor, for example, drives anti-survival RHOA/JNK activation, suggesting novel candidate biomarkers of rocaglate clinical outcomes. Translation-machinery analysis by MATRIX identifed rocaglate-induced dependence on specific translation factors including eEF1ϵ1 that drive remodeling. Novel rocaglate RNA-binding targets revealed by clampome studies remain under detailed evaluation as mediators of drug activities. Discussion: Our original proteome-level interrogation revealed that the complete cellular response to these historical “translation inhibitors” is mediated by comprehensive translational landscape remodeling. Effects on a broader suite of RNA binding proteins than eIF4A1 alone we suggest mediate the potent antitumor activities of these unique compounds, elucidation of which permits development of novel precision approaches to targeted translational deregulation in cancer. Citation Format: Tyler A. Cunningham, J. J. David Ho, Paola Manara, Stephen Lee, Jonathan H. Schatz. Proteomic approaches define rocaglates as translation remodelers with multiple protein targets [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P028.

A cold shock protein promotes high-temperature microbial growth through binding to diverse RNA species

Endowing mesophilic microorganisms with high-temperature resistance is highly desirable for industrial microbial fermentation. Here, we report a cold-shock protein (CspL) that is an RNA chaperone protein from a lactate producing thermophile strain (Bacillus coagulans 2–6), which is able to recombinantly confer strong high-temperature resistance to other microorganisms. Transgenic cspL expression massively enhanced high-temperature growth of Escherichia coli (a 2.4-fold biomass increase at 45 °C) and eukaryote Saccharomyces cerevisiae (a 2.6-fold biomass increase at 36 °C). Importantly, we also found that CspL promotes growth rates at normal temperatures. Mechanistically, bio-layer interferometry characterized CspL’s nucleotide-binding functions in vitro, while in vivo we used RNA-Seq and RIP-Seq to reveal CspL’s global effects on mRNA accumulation and CspL’s direct RNA binding targets, respectively. Thus, beyond establishing how a cold-shock protein chaperone provides high-temperature resistance, our study introduces a strategy that may facilitate industrial thermal fermentation.

A transgenic DND1GFP fusion allele reports in vivo expression and RNA-binding targets in undifferentiated mouse germ cells†.

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

13P The identification of binding targets for pro-metastatic protein NIFK in lung cancer

Background: We previously identified nucleolar protein interacting with the FHA domain of pKi-67 (NIFK) as a poor prognostic marker in lung cancer. Furthermore, NIFK overexpression promotes cancer metastasis via decreasing casein kinase 1α (CK1α) expression, a suppressor of pro-metastatic TCF4/β-catenin signaling. We further observe the axis is induced by the downregulation of a novel transcription factor RUNX1. However, the interplay between NIFK, CK1α and RUNX1 remains largely unknown. In this study, we aim to unravel the interactive mechanism of the signaling axis NIFK/RUNX1/CK1α, and to characterize its potential clinical value in cancer patient cohort. Methods: NIFK is pointed out to possess RNA binding function via the RNA Recognition Motif (RRM). RNA immunoprecipitation assay was performed using specific NIFK antibody in lung cancer PC13 and A549 cells after NIFK overexpression. RNA targets were analyzed by RT-PCR and Next Generation Sequencing (NGS). In addition, relative NIFK, RUNX1 and CK1α expression and its association with patient survival outcome were investigated in lung cancer cohorts. Results: We observe the increase in amount of RNA binding targets after NIFK overexpression respectively in PC13 and A549 cells as comparing with the control. Moreover, NIFK appears to bind with RUNX1 RNA but not CK1α suggesting it is the initial step of NIFK-mediated TCF4/β-catenin signaling regulation. NIFK also leads to RUNX1 RNA instability of which mechanism remains to be explored. RUNX1 is observed to associate with good prognosis in lung cancer datasets (jacob-00182-CANDF and GSE13213). In addition, relative expression of the axis is further revealed in lung cancer cohort. Conclusions: We first show the interactive mechanism of pro-metastatic molecule NIFK in regulating TCF4/β-catenin signaling, which might contribute to therapeutic target design for lung cancer patients. Legal entity responsible for the study: Chang Gung Memorial Hospital Funding: Ministry of Science and Technology, Taiwan Disclosure: All authors have declared no conflicts of interest.

Shep regulates Drosophila neuronal remodeling by controlling transcription of its chromatin targets.

Neuronal remodeling is crucial for formation of the mature nervous system and disruption of this process can lead to neuropsychiatric diseases. Global gene expression changes in neurons during remodeling as well as the factors that regulate these changes remain poorly defined. To elucidate this process, we performed RNA-seq on isolated Drosophila larval and pupal neurons and found upregulated synaptic signaling and downregulated gene expression regulators as a result of normal neuronal metamorphosis. We further tested the role of alan shepard (shep), which encodes an evolutionarily conserved RNA-binding protein required for proper neuronal remodeling. Depletion of shep in neurons prevents the execution of metamorphic gene expression patterns, and shep-regulated genes correspond to Shep chromatin and/or RNA-binding targets. Reduced expression of a Shep-inhibited target gene that we identified, brat, is sufficient to rescue neuronal remodeling defects of shep knockdown flies. Our results reveal direct regulation of transcriptional programs by Shep to regulate neuronal remodeling during metamorphosis.

Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80% of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.

RNA binding protein Musashi-1 directly targets Msi2 and Erh during early testis germ cell development and interacts with IPO5 upon translocation to the nucleus.

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in Drosophila and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic Msi1 testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, Msi2 and Erh, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function.

Asymptotic Number of Hairpins of Saturated RNA Secondary Structures

In the absence of chaperone molecules, RNA folding is believed to depend on the distribution of kinetic traps in the energy landscape of all secondary structures. Kinetic traps in the Nussinov energy model are precisely those secondary structures that are saturated, meaning that no base pair can be added without introducing either a pseudoknot or base triple. In this paper, we compute the asymptotic expected number of hairpins in saturated structures. For instance, if every hairpin is required to contain at least θ=3 unpaired bases and the probability that any two positions can base-pair is p=3/8, then the asymptotic number of saturated structures is 1.34685[Symbol: see text]n (-3/2)[Symbol: see text]1.62178 (n) , and the asymptotic expected number of hairpins follows a normal distribution with mean [Formula: see text]. Similar results are given for values θ=1,3, and p=1,1/2,3/8; for instance, when θ=1 and p=1, the asymptotic expected number of hairpins in saturated secondary structures is 0.123194[Symbol: see text]n, a value greater than the asymptotic expected number 0.105573[Symbol: see text]n of hairpins over all secondary structures. Since RNA binding targets are often found in hairpin regions, it follows that saturated structures present potentially more binding targets than nonsaturated structures, on average. Next, we describe a novel algorithm to compute the hairpin profile of a given RNA sequence: given RNA sequence a 1,…,a n , for each integer k, we compute that secondary structure S k having minimum energy in the Nussinov energy model, taken over all secondary structures having k hairpins. We expect that an extension of our algorithm to the Turner energy model may provide more accurate structure prediction for particular RNAs, such as tRNAs and purine riboswitches, known to have a particular number of hairpins. Mathematica(™) computations, C and Python source code, and additional supplementary information are available at the website http://bioinformatics.bc.edu/clotelab/RNAhairpinProfile/ .

Direct RNA Target Analysis Identifies Signatures That Define Msi2's Role in Sustaining HSC Cell Fate.

AbstractAbstract 2324Hematopoietic stem cells (HSC) must maintain normal cell fate decisions between symmetric and asymmetric divisions as alterations can lead to hematopoietic malignancies. The MSI2 RNA binding protein is upregulated in patients with a poor clinical prognosis in acute myeloid leukemia and in the blast crisis phase of chronic myelogenous leukemia. The related RNA-binding protein Msi1 has been shown to block translation of specific target mRNAs by interacting with the 3'UTR. To understand the role of Msi2 in both normal and leukemic contexts, we are characterizing mice with a conditional deletion of Msi2 in the hematopoietic compartment. Msi2 conditional knockouts have reduced overall number of HSCs in the bone marrow and have decreased engraftment capacity in congenic transplants, however the molecular mechanisms through which Msi2 elicits these effects remains unclear.To understand how Msi2 alters HSC self renewal, we utilized Mx1-Cre::Msi2flox/flox conditional mice, enabling Msi2 inactivation via poly(I):poly(C) injection. We first tested if Msi2 deleted HSCs or MPPs had an intrinsic defect in cell fate decision and proliferation. We performed proliferation and colony assays on sorted HSCs and MPPs and found reduced cellular numbers in both the HSCs and MPPs with an increase in the frequency of more differentiated cells based on Mac/Gr1 staining after seven days. These reduced overall cell numbers in vitro may have resulted from a defect in maintaining the stem cell population or a decrease in proliferative capacity. To assess if there was a defect in the initial HSC cell division, we examined Numb protein levels and distribution as a surrogate readout for asymmetric cell division. Numb mRNA is a known target of Msi binding and translational repression. Surprisingly, in the Msi2 null HSC or MPPs we observed no difference in the overall staining of Numb indicating that Msi2 deficiency does not globally alter Numb levels. However, we did observe a decrease in the percentage of cells that underwent asymmetric Numb segregation in the MPPs and an increase in the percentage of cells that showed increased Numb staining in some of the daughter pairs in both HSCs and MPPs, indicating increased commitment away from the hematopoietic stem and progenitors.Although controversial, Notch signaling has been implicated in self renewal of HSCs and as a critical downstream target of the Msi family through Numb inactivation. However, the Notch signaling pathway was not noted to be significantly altered in gene set analysis from microarrays performed on Msi2 deleted HSC enriched populations. Moreover using quantitative PCR for Notch target genes in sorted Linlow, c-kit+ and Sca+(LSK) cells we found no statistical difference in the expression of Notch targets (Notch1, Notch2, Hes1 or Myc). These interesting negative results prompted us take a more global approach in characterizing the direct targets of Msi2 in hematopoietic cells.Due to the requirement for a large number of cells, we utilized K562 cells overexpressing a FLAG-tagged version of MSI2 to identify its direct RNA-binding targets. Using recently developed technologies, we performed experiments with UV-cross-linked and immunoprecipitated MSI2 which was then followed by RNA-sequencing (HITS-CLIP) to identify the global direct binding. Our analysis characterized the distribution of binding across the genome. Additionally, gene set enrichment analysis (GSEA) indicated a positive correlation of genes that were upregulated in the Msi2 deleted LSKs and the top 3-fold bound RNA targets (2,713 genes). Using the entire RNA target list we identified gene set signatures including “Cell Cycle”, “Self-renewal” and “HSC to CMP” that matched our results in the microarray from Msi2 deleted LSKs.In summary, direct RNA target analysis for MSI2 in human leukemia cells overlapped with self renewal and differentiation gene sets in mouse HSC enriched populations and correlated with the phenotypes we observed in isolated HSCs grown in vitro lacking Msi2. These results uncover the complexity of MSI2 RNA binding targets and have important implications for both normal and leukemic stem cell biology. Disclosures:Ebert:Celgene: Consultancy; Genoptix: Consultancy.

TDP-43 and FUS/TLS yield a target-rich haul in ALS

Genome-wide approaches are used to discover the RNA-binding targets of the amyotrophic lateral sclerosis (ALS) disease protein FUS/TLS. These studies reveal some shared targets with another ALS-linked RNA-binding protein, TDP-43, suggesting common pathogenic mechanisms.

Structure Function Analysis of an ADP-ribosyltransferase Type III Effector and Its RNA-binding Target in Plant Immunity

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.7-Å resolution reveals two unique protruding loops, L1 and L4, not found in other mono-ADP-ribosyltransferases. Site-directed mutagenesis demonstrates that these loops are essential for substrate recognition and enzymatic activity. HopU1 ADP-ribosylates the conserved arginine 49 of GRP7, and this reduces the ability of GRP7 to bind RNA in vitro. In vivo, expression of GRP7 with Arg-49 replaced with lysine does not complement the reduced immune responses of the Arabidopsis thaliana grp7-1 mutant demonstrating the importance of this residue for GRP7 function. These data provide mechanistic details how HopU1 recognizes this novel type of substrate and highlights the role of GRP7 in plant immunity.

RNA Binding Targets Aminoacyl-tRNA Synthetases to Translating Ribosomes

Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to poxvirus or alphavirus factories in infected cells. Immunoproximity fluorescence indicates close proximity between MSC and the ribosome. Stress induced-translational shutdown recruits the MSC to stress-granules, a depot for mRNA and translation components. MSC binding to mRNA provides a facile explanation for its delivery to translating ribosomes and stress granules. These findings, along with the abundance of the MSC (9 × 10(6) copies per cell, roughly equimolar with ribosomes), is consistent with the idea that MSC specificity, recently reported to vary with cellular stress (Netzer, N., Goodenbour, J. M., David, A., Dittmar, K. A., Jones, R. B., Schneider, J. R., Boone, D., Eves, E. M., Rosner, M. R., Gibbs, J. S., Embry, A., Dolan, B., Das, S., Hickman, H. D., Berglund, P., Bennink, J. R., Yewdell, J. W., and Pan, T. (2009) Nature 462, 522-526) can be modulated at the level of individual mRNAs to modify decoding of specific gene products.

2.111 RNA binding targets of the recessive parkinsonism protein DJ-1 reveals involvement in mitochondrial, oxidative stress and PTEN/Akt survival pathways

2.111 RNA binding targets of the recessive parkinsonism protein DJ-1 reveals involvement in mitochondrial, oxidative stress and PTEN/Akt survival pathways

Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets

StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors.

Characterization of the Pre-mRNA Binding Site for Yeast Ribosomal Protein L32: The Importance of a Purine-rich Internal Loop

The structure of the RNA binding target for Saccharomyces cerevisiaeribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5′ end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G·U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.