Recognition Of Poly Research Articles

A PARylation-phosphorylation cascade promotes TOPBP1 loading and RPA-RAD51 exchange in homologous recombination

The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to chemotherapy. While many factors have been functionally characterized in HR, such as TOPBP1, their precise regulation remains unclear. Here, we report that TOPBP1 interacts with the RNA-binding protein HTATSF1 in a cell-cycle- and phosphorylation-dependent manner. Mechanistically, CK2 phosphorylates HTATSF1 to facilitate binding to TOPBP1, which promotes S-phase-specific TOPBP1 recruitment to damaged chromatin and subsequent RPA/RAD51-dependent HR, genome integrity, and cancer-cell viability. The localization of HTATSF1-TOPBP1 to DSBs is potentially independent of the transcription-coupled RNA-binding and processing capacity of HTATSF1 but rather relies on the recognition of poly(ADP-ribosyl)ated RPA by HTATSF1, which can be blunted with PARP inhibitors. Together, our study provides a mechanistic insight into TOPBP1 loading at HR-prone DSB sites via HTATSF1 and reveals how RPA-RAD51 exchange is tuned by a PARylation-phosphorylation cascade.

Poly I:C stimulation in-vitro as a marker for an antiviral response in different cell types generated from Buffalo (Bubalus bubalis)

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNβ and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNβ expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNβ production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNβ expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-β expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNβ expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.

Innate immune responses through Toll-like receptor 3 require human-antigen-R-mediated Atp6v0d2 mRNA stabilization

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA derived from virus and its synthetic analogue, polyinosinic–polycytidylic acid [poly(I:C)]. Upon poly(I:C) binding, TLR3 activates transcription factors to express inflammatory cytokines and type I interferon. TLR3 is located in the endosomes and its recognition of poly(I:C) and activation of downstream signaling is regulated by endosomal acidification. However, the mechanism of post-transcriptional regulation in TLR3-mediated innate responses remains unclear. Here, we focused on Human antigen R (HuR, also known as ELAVL1) that recognizes and binds to the 3′ untranslated regions (3′UTRs) of target mRNAs, thereby protecting them from mRNA degradation, and found that HuR-deficient murine macrophage cells showed significantly reduced Ifnb1 mRNA expression after poly(I:C) stimulation. HuR-deficient cells also showed a marked reduction in the expression of Atp6v0d2 mRNA, which encodes a subunit of vacuolar-type H+ ATPase (V-ATPase), and therefore reduced endosomal acidification. HuR associated with the 3′UTR of Atp6v0d2 mRNA and the stability of Atp6v0d2 mRNA was maintained by its association with HuR. Taken together, our results suggest that HuR stabilizes Atp6v0d2 mRNA, which is required for the TLR3-mediated innate immune responses.

Recognition of Poly(A) RNA through Its Intrinsic Helical Structure.

The polyadenosine (poly(A)) tail, which is found on the 3’ end of almost all eukaryotic messenger RNAs (mRNAs), plays an important role in the posttranscriptional regulation of gene expression. Shortening of the poly(A) tail, a process known as deadenylation, is thought to be the first and rate-limiting step of mRNA turnover. Deadenylation is performed by the Pan2–Pan3 and Ccr4–Not complexes that contain highly conserved exonuclease enzymes Pan2, and Ccr4 and Caf1, respectively. These complexes have been extensively studied, but the mechanisms of how the deadenylase enzymes recognize the poly(A) tail were poorly understood until recently. Here, we summarize recent work from our laboratory demonstrating that the highly conserved Pan2 exonuclease recognizes the poly(A) tail, not through adenine-specific functional groups, but through the conformation of poly(A) RNA. Our biochemical, biophysical, and structural investigations suggest that poly(A) forms an intrinsic base-stacked, single-stranded helical conformation that is recognized by Pan2, and that disruption of this structure inhibits both Pan2 and Caf1. This intrinsic structure has been shown to be important in poly(A) recognition in other biological processes, further underlining the importance of the unique conformation of poly(A).

Pyrrolocytosine–pyrene conjugates as fluorescent and CD probes for the fine sensing of ds-polynucleotide secondary structure and specific recognition of poly G

Multipurpose dual method (fluorescence and CD) probes differentiate between various ds-RNA/RNA structures and specifically recognise Watson–Crick pairing complementary poly G.

Mechanisms of mRNA polyadenylation

mRNA 3'-end processing involves the addition of a poly(A) tail based on the recognition of the poly(A) signal and subsequent cleavage of the mRNA at the poly(A) site. Alternative polyadenylation (APA) is emerging as a novel mechanism of gene expression regulation in normal and in disease states. APA results from the recognition of less canonical proximal or distal poly(A) signals leading to changes in the 3' untranslated region (UTR) lengths and even in some cases changes in the coding sequence of the distal part of the transcript. Consequently, RNA-binding proteins and/or microRNAs may differentially bind to shorter or longer isoforms. These changes may eventually alter the stability, localization, and/or translational efficiency of the mRNAs. Overall, the 3' UTRs are gaining more attention as they possess a significant posttranscriptional regulation potential guided by APA, microRNAs, and RNA-binding proteins. Here we provide an overview of the recent developments in the APA field in connection with cancer as a potential oncogene activator and/or tumor suppressor silencing mechanism. A better understanding of the extent and significance of APA deregulation will pave the way to possible new developments to utilize the APA machinery and its downstream effects in cancer cells for diagnostic and therapeutic applications.

Breaking the protein-RNA recognition code

Breaking the protein-RNA recognition code

A novel genome-wide polyadenylation sites recognition system based on condition random field.

Polyadenylation including the cleavage of pre-mRNA and addition of a stretch of adenosines to the 3'-end is an essential step of pre-mRNA processing in eukayotes. The known regulatory role of polyadenylation in mRNA localization, stability, and translation and the emerging link between poly(A) and disease states underline the necessary to fully characterize polyadenylation sites. Several artificial intelligence methods have been proposed for poly(A) sites recognition. However, these methods are suitable to small subsets of genome sequences. It is necessary to propose a method for genome-wide recognition of poly(A) sites. Recent efforts have found a lot of poly(A) related factors on DNA level. Here, we proposed a novel genome-wide poly(A) recognition method based on the Condition Random Field (CRF) by integrating multiple features. Compared with the polya_svm (the most accurate program for prediction of poly(A) sites till date), our method had a higher performance with the area under ROC curve(0.8621 versus 0.6796). The result suggests that our method is an effective method in genome wide poly(A) sites recognition.

Comprehensive recognition of messenger RNA polyadenylation patterns in plants

The polyadenylation of messenger RNA (mRNA) in eukaryotes is an essential step in gene expression. Currently, with the in-depth sequencing, a considerable amount of alternative poly(A) sites have been found in the coding sequences and introns, while there was little study on these unconventional poly(A) sites and their signals. To study the signals of mRNA polyadenylation, an effective poly(A) signal pattern recognition model was established to select and analyze the nucleotide patterns in the poly(A) siterelated regions from large scale sequences generated from Sanger and next generation sequencing technologies. Our model, integrating a pattern and an assembly analysis pipelines and several visualization methods could be applied to various species. Through recognition of poly(A) patterns in three species including rice, Arabidopsis and Chlamydomonas reinhardtii , the experimental results showed that this model was able to select effective poly(A) signal patterns for poly(A) sites and alternative poly(A) sites to compare the poly(A) signals in different species and different regions, and to enhance the accuracy of poly(A) sites recognition to a larger extent.

Recognition of poly(dimethylsiloxane) with phage displayed peptides

We demonstrate for the first time a powerful yet benign approach for identification of binding motifs to poly(dimethylsiloxane) (PDMS) via comprehensively screened phage displayed peptides. Our results show that PDMS can be selectively recognized with peptide-displaying phages and bifunctional peptides. Further, along with the development of PDMS-based microstructures, recognition of PDMS with phage displayed peptides can be specifically localized in these microstructures.

Dragon PolyA Spotter: predictor of poly(A) motifs within human genomic DNA sequences

Motivation: Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants.Contact: vladimir.bajic@kaust.edu.saSupplementary information: Supplementary data are available at Bioinformatics online.

Unique Properties of the Mtr4p−Poly(A) Complex Suggest a Role in Substrate Targeting

Mtr4p is a DEVH-box helicase required for 3'-end processing and degradation of various nuclear RNA substrates. In particular, Mtr4p is essential for the creation of 5.8S rRNA, U4 snRNA, and some snoRNAs and for the degradation of cryptic unstable transcripts (CUTs), aberrant mRNAs, and aberrant tRNAs. Many instances of 3'-end processing require limited polyadenylation to proceed. While polyadenylation can signal degradation in species from bacteria to humans, the mechanism whereby polyadenylated substrates are delivered to the degradation machinery is unknown. Our previous work has shown that Mtr4p preferentially binds poly(A) RNA. We suspect that this preference aids in targeting polyadenylated RNAs to the exosome. In these studies, we have investigated the mechanism underlying the preference of Mtr4p for poly(A) substrates as a means of understanding how Mtr4p might facilitate targeting. Our analysis has revealed that recognition of poly(A) substrates involves sequence-specific changes in the architecture of Mtr4p-RNA complexes. Furthermore, these differences significantly affect downstream activities. In particular, homopolymeric stretches like poly(A) ineffectively stimulate the ATPase activity of Mtr4p and suppress the rate of dissociation of the Mtr4p-RNA complex. These findings indicate that the Mtr4p-poly(A) complex is unique and ideally suited for targeting key substrates to the exosome.

Crystal Structure of the First KH Domain of Human Poly(C)-binding Protein-2 in Complex with a C-rich Strand of Human Telomeric DNA at 1.7 Å

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation.

Cleavage and polyadenylation factor (CPF) is a multi-protein complex that functions in pre-mRNA 3'-end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3'-end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3'-end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C-terminal domain (CTD) of RNAP II plays a major role in coupling 3'-end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

The Cleavage/Polyadenylation Activity Triggered by a U-rich Motif Sequence Is Differently Required Depending on the Poly(A) Site Location at Either the First or Last 3′-Terminal Exon of the 2′-5′ Oligo(A) Synthetase Gene

Production of the two mRNAs encoding distinct forms of 2'-5'-oligoadenylate synthetase depends on processing that involves the recognition of alternative poly(A) sites and an internal 5'-splice site located within the first 3'-terminal exon. The resulting 1.6- and 1.8-kb mRNAs are expressed in fibroblast cell lines, whereas lymphoblastoid B cells, such as Daudi, produce only the 1.8-kb mRNA. In the present study, we have shown that the 3'-end processing at the last 3'-terminal exon occurs independently of the core poly(A) site sequence or the presence of regulatory elements. In contrast, in Daudi cells, the recognition of the poly(A) site at the first 3'-terminal exon is impaired because of an unfavorable sequence context. The 3'-end processing at this particular location requires a strong stabilization of the cleavage/polyadenylation factors, which can be achieved by the insertion of a 25-nucleotide long U-rich motif identified upstream of the last poly(A) site. Consequently, we speculate that in cells expressing the 1.6-kb mRNA, such as fibroblasts, direct or indirect participation of a specific mechanism or cell type-specific factors are required for an efficient polyadenylation at the first 3'-terminal exon.

Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage and polyadenylation factor.

Recognition of poly(A) sites in yeast pre-mRNAs is poorly understood. Employing an in vitro cleavage system with cleavage and polyadenylation factor (CPF) and cleavage factor IA we show that the efficiency and positioning elements are dispensable for poly(A)-site recognition within a short CYC1 substrate in vitro. Instead, U-rich elements immediately upstream and downstream of the poly(A) site mediate cleavage-site recognition within CYC1 and ADH1 pre-mRNAs. These elements act in concert with the poly(A) site to produce multiple recognition sites for the processing machinery, since combinations of mutations within these elements were most effective in cleavage inhibition. Intriguingly, introduction of a U-rich element downstream of the GAL7 poly(A) site strongly enhanced cleavage, underscoring the importance of downstream sequences in general. RNA- binding analyses demonstrate that cleavage depends on the recognition of the poly(A)-site region by CPF. Consistent with in vitro results, mutation of sequences upstream and downstream of the poly(A) site affected 3'-end formation in vivo. A model for yeast pre-mRNA cleavage-site recognition outlines an unanticipated high conservation of yeast and mammalian 3'-end processing mechanisms.

Specificity of guanylic RNases to polynucleotide substrates

Kinetic parameters kcat and Km were measured for cleavage of poly I, poly A, poly U, poly C and poly I. poly C by guanyl-specific RNases Sa, Pb1 and T1 and compared with that of guanyl-preferential RNase Bi. Catalytic efficiencies of the investigated enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi.

Chain-length recognition of poly(methacrylic acids) by a monolayer of a poly(oxyethylene)-based amphiphile at the air—water interface

Formation of monolayers of a poly(oxyethylene) segment containing amphiphiles on water and their chain-length recognition of a guest poly(methacrylic acid) in the subphase are described.