Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.     

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K_d’s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K_d’s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.     

G-quadruplex conformation and dynamics are determined by loop length and sequence

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.     

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.     

Analysis of nucleic acid binding by a recombinant translin-trax complex

Translin is a highly conserved mammalian RNA and DNA-binding protein involved in DNA recombination and RNA trafficking. Crystal structures of mouse and human translin have been solved, but do not provide information about nucleic acid binding or recognition. Translin has a partner protein, translin-associated factor x (trax), which is believed to regulate translin’s subcellular locale and affinity for certain RNA and DNA sequences. Here we present a comparative study of recombinant translin and translin-trax complex binding to specific RNA and DNA sequences. It was observed that translin preferentially binds to G-rich RNA sequences whereas translin-trax preferentially binds G-rich DNA sequences. Translin can bind mRNA sequences with sub-micromolar K_d values, and the complex with trax can bind G-rich DNA with similar affinity. We conclude that trax acts to regulate translin’s RNA and DNA binding affinities as part of a cellular RNA trafficking mechanism.     

基于G4-配体的DNA模拟酶在生物传感中的发展与应用

鸟苷酸-四联体DNA (G4 DNA)是由富含串联重复鸟苷酸(G)的DNA或RNA序列形成的G4片层,并堆叠而成一类独特的核酸二级结构。G4 DNA结合多种特异性配体可形成具有催化过氧化氢活性的G4 DNA模拟酶(G4 DNAzyme)。由于G4 DNAzyme存在着序列构成简便灵活、适合多样传感平台检测等特点,其在新型生物传感方法研发、医学检测新技术研究等领域中应用前景广阔。本文主要依据G4 DNA结合配体的不同,对近年来新发展出的G4 DNAzyme进行分类与回顾,归纳为含氯化血红素(hemin)的G4-Hemin DNAzyme与非G4-Hemin DNAzyme。前者是目前G4 DNA模拟酶的研究主流——本文主要归纳了G4-Hemin DNAzyme在金属阳离子、生物小分子及生物大分子的检测分析方向上所取得的重要研究进展,并阐述其在生物传感领域的影响和优势;后者中的配体则主要包括金属阳离子N-甲基吗啡啉(4-methylmorpholine, NMM)、硫磺素T(thioflavin T,ThT)及新型金属配体(Cu²⁺Ce³⁺)等。...     

Analysis of Nuclear Export Sequence Regions of FUS-Related RNA-Binding Proteins in Essential Tremor

Background and Objective

Genes encoding RNA-binding proteins, including FUS and TDP43, play a central role in different neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Recently, a mutation located in the nuclear export signal (NES) of the FUS gene has been reported to cause an autosomal dominant form of familial Essential tremor.

Material and Methods

We sequenced the exons coding the NES domains of five RNA-binding proteins (TARDBP, hnRNPA2B1, hnRNPA1, TAF15 and EWSR1) that have been previously implicated in neurodegeneration in a series of 257 essential tremor (ET) cases and 376 healthy controls. We genotyped 404 additional ET subjects and 510 healthy controls to assess the frequency of the EWSR1 p.R471C substitution.

Results

We identified a rare EWSR1 p.R471C substitution, which is highly conserved, in a single subject with familial ET. The pathogenicity of this substitution remains equivocal, as DNA samples from relatives were not available and the genotyping of 404 additional ET subjects did not reveal any further carriers. No other variants were observed with significant allele frequency differences compared to controls in the NES coding regions.

Conclusions

The present study demonstrates that the NES domains of RNA-binding proteins are highly conserved. The role of the EWSR1 p.R471C substitution needs to be further evaluated in future studies.     

EWSR1, a multifunctional protein,regulates cellular function and aging via genetic and epigenetic pathways

Ewing's sarcoma (EWS) is a bone cancer arising predominantly in young children. EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) gene is ubiquitously expressed in most cell types, indicating it has diverse roles in various cellular processes and organ development. Recently, several studies have shown that missense mutations of EWSR1 genes are known to be associated with central nervous system disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Otherwise, EWSR1 plays epigenetic roles in gene expression, RNA processing, and cellular signal transduction. Interestingly, EWSR1 controls micro RNA (miRNA) levels via Drosha, leading to autophagy dysfunction and impaired dermal development. Ewsr1 deficiency also leads to premature senescence of blood cells and gamete cells with a high rate of apoptosis due to the abnormal meiosis. Despite these roles of EWSR1 in various cellular functions, the exact mechanisms are not yet understood. In this context, the current review overviews a large body of evidence and discusses on what EWSR1 genetic mutations are associated with brain diseases and on how EWSR1 modulates cellular function via the epigenetic pathway. This will provide a better understanding of bona fide roles of EWSR1 in aging and its association with brain disorders.     

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the ‘flanking effect’ in this work. This ‘flanking effect’ was more pronounced when nucleotides were added at the 5′-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5′-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.     

An RNA-binding Protein,Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.     

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process.     

A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila

Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate’s somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.     

Characterization of RNA-binding properties of three types of RNA-binding proteins in Anabaena sp. PPC 7120.

The Rbp proteins in cyanobacteria are RNA-binding proteins with a single RNA recognition motif or RRM. A comprehensive assembly of genomic data suggests that there are two major classes of Rbp proteins (classes I and II) that diverged before the diversification of cyanobacteria. Class I proteins are further classified into two types with or without a C-terminal glycine-rich domain. The results of selection from a random RNA pool suggest that RbpA1 (class I) has affinity to C-rich and G-rich sequences. In vitro RNA binding assay with homopolymers indicated that class II protein has low affinity to poly(G) in contrast with class I proteins. Site-specific mutagenesis analysis of the RRM in RbpA1 showed that the aromatic residues Tyr4 or Phe46 are important in RNA binding as well as maintenance of secondary structure. We also tested various truncated proteins lacking the C-terminal domain as well as point mutants. Most of these proteins exhibited decreased affinity to RNA. Circular dichroism analysis as well as chromatographic analysis showed that Tyr4 and Phe46 are also important in maintaining the structure of RbpA1 protein. The C-terminal glycine-rich domain itself does not contribute much to the RNA-binding, but Arg83 which is located close to the C-terminal end of RRM is important in the RNA-binding.     

The sequence selectivity of KSRP explains its flexibility in the recognition of the RNA targets

K-homology (KH) splicing regulator protein (KSRP) is a multi-domain RNA-binding protein that regulates different steps of mRNA metabolism, from mRNA splicing to mRNA decay, interacting with a broad range of RNA sequences. To understand how KSRP recognizes its different RNA targets it is necessary to define the general rules of KSRP–RNA interaction. We describe here a complete scaffold-independent analysis of the RNA-binding potential of the four KH domains of KSRP. The analysis shows that KH3 binds to the RNA with a significantly higher affinity than the other domains and recognizes specifically a G-rich target. It also demonstrates that the other KH domains of KSRP display different sequence preferences explaining the broad range of targets recognized by the protein. Further, KSRP shows a strong negative selectivity for sequences containing several adjacent Cytosines limiting the target choice of KSRP within single-stranded RNA regions. The in-depth analysis of the RNA-binding potential of the KH domains of KSRP provides us with an understanding of the role of low sequence specificity domains in RNA recognition by multi-domain RNA-binding proteins.     

Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPs in vivo

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F•mRNA•PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP–eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4G•mRNA•PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G–mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP–eIF4G association is only one of several interactions that stabilize eIF4F•mRNA complexes, and emphasize that closed-loop mRNP formation via PABP–eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F•mRNA assembly.