Structural Adaptation in Its Orphan Domain Engenders Betaglycan with an Alternate Mode of Growth Factor Binding Relative to Endoglin.

Betaglycan (BG) and endoglin (ENG), homologous co-receptors of the TGF-ß family, potentiate the signaling activity of TGF-ß2 and inhibin A, and BMP-9 and BMP-10, respectively. BG exists as monomer and forms 1:1 growth factor (GF) complexes, while ENG exists as a dimer and forms 2:1 GF complexes. Herein, the structure of the BG orphan domain (BGO) reveals an insertion that blocks the region that the endoglin orphan domain (ENGO) uses to bind BMP-9, preventing it from binding in the same manner. Using binding studies with domain-deleted forms of TGF-ß and BGO, as well as small-angle X-ray scattering data, BGO is shown to bind its cognate GF in an entirely different manner compared with ENGO. The alternative interfaces likely engender BG and ENG with the ability to selectively bind and target their cognate GFs in a unique temporal-spatial manner, without interfering with one another or other TGF-ß family GFs.

TGF-ß2 uses the concave surface of its extended finger region to bind betaglycan's ZP domain via three residues specific to TGF-ß and inhibin-α.

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-ß family that selectively binds transforming growth factor-ß (TGF-ß) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo Here, using NMR titrations of methyl-labeled TGF-ß2 with BG's C-terminal binding domain, BGZP-C, and surface plasmon resonance binding measurements with TGF-ß2 variants, we found that the BGZP-C-binding site on TGF-ß2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-ß isoforms and the InhA α-subunit, but they are unconserved in other TGF-ß family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-ß2 variants with the corresponding residues from BMP-2 bound BGZP-C more weakly than corresponding alanine variants. The BGZP-C-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-ß2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-ß2 and InhA bind BGZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BGZP-C-binding site on TGF-ß2 and shed light on the specificity of BG for select TGF-ß-type GFs and the mechanisms by which BG influences their signaling.

A Novel TGFß Trap Blocks Chemotherapeutics-Induced TGFß1 Signaling and Enhances Their Anticancer Activity in Gynecologic Cancers.

Purpose: We investigated the mechanisms of how TGFß pathway is activated by chemotherapeutics and whether a novel TGFß trap called RER can block chemotherapeutics-induced TGFß pathway activation and enhance their antitumor activity in gynecologic cancer.Patients and Methods: An unbiased bioinformatic analysis of differentially expressed genes in 31 ovarian cases due to chemotherapy was used to identify altered master regulators. Phosphorylated Smad2 was determined in 30 paired cervical cancer using IHC. Furthermore, the effects of chemotherapeutics on TGFß signaling and function, and the effects of RER on chemotherapy-induced TGFß signaling were determined in gynecologic cancer cells.Results: Chemotherapy-induced transcriptome alteration in ovarian cancer was significantly associated with TGFß signaling activation. Chemotherapy was found to activate TGFß signaling as indicated by phosphorylated Smad2 in paired cervical tumor samples (pre- and post-chemotherapy). Similar to TGFß1, chemotherapeutics were found to stimulate Smad2/3 phosphorylation, cell migration, and markers related to epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC). These TGFß-like effects were due to the stimulation of TGFß1 expression and secretion, and could all be abrogated by TGFß inhibitors including a novel TGFß trap protein called RER both in vitro and in vivo Importantly, combination treatment with RER and cisplatin showed a higher tumor inhibitory activity than either agent alone in a xenograft model of ovarian cancer.Conclusions: Chemotherapeutics can stimulate TGFß1 production and consequently enhance TGFß signaling, EMT, and CSC features resulting in reduced chemo-sensitivity. Combination therapy with a TGFß inhibitor should alleviate this unintended side effect of chemotherapeutics and enhance their therapeutic efficacy. Clin Cancer Res; 24(12); 2780-93. ©2018 AACR.

Correction: A novel highly potent trivalent TGF-ß receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

[This corrects the article DOI: 10.18632/oncotarget.13343.].

RNAcentral: a comprehensive database of non-coding RNA sequences.

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.

A novel highly potent trivalent TGF-ß receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

The effects of transforming growth factor beta (TGF-ß) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-ß functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-ß pathway, we investigated the effect of systemic treatment with a TGF-ß inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-ß receptor trap, RER, comprised of domains derived from the TGF-ß type II and type III receptors. This trap was shown to completely block TßRII binding, to antagonize TGF-ß1 and TGF-ß3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-ß activities than a pan TGF-ß neutralizing antibody and a TGF-ß receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-ß acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-ß signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

RNAcentral: an international database of ncRNA sequences.

The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis.

BACKGROUND: Trans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic degradation. Comparative sequence analysis of bacterial tmRNAs provides considerable insights into their secondary structures in which a tRNA-like domain and an mRNA-like region are connected by a variable number of pseudoknots. Progress toward understanding the molecular mechanism of trans-translation is hampered by our limited knowledge about the structure of tmRNA:SmpB complexes. RESULTS: Complexes consisting of M. tuberculosis tmRNA and E. coli SmpB tag truncated proteins poorly in E. coli. In contrast, the tagging activity of E. coli tmRNA is well supported by M. tuberculosis SmpB that is expressed in E. coli. To investigate this incompatibility, we constructed 12 chimeric tmRNA molecules composed of structural features derived from both E. coli and M. tuberculosis. Our studies demonstrate that replacing the hp5-pk2-pk3-pk4 segment of E. coli tmRNA with the equivalent segment of M. tuberculosis tmRNA has no significant effect on the tagging efficiency of chimeric tmRNAs in the presence of E. coli SmpB. Replacing either helices 2b-2d, the single-stranded part of the ORF, pk1, or residues 79-89 of E. coli tmRNA with the equivalent features of M. tuberculosis tmRNA yields chimeric tmRNAs that are tagged at 68 to 88 percent of what is observed with E. coli tmRNA. Exchanging segments composed of either pk1 and the single-stranded segment upstream of the ORF or helices 2b-2d and pk1 results in markedly impaired tagging activity. CONCLUSION: Our observations demonstrate the existence of functionally important but as yet uncharacterized structural constraints in the segment of tmRNA that connects its TLD to the ORF used for resuming translation. As trans-translation is important for the survival of M. tuberculosis, our work provides a new target for pharmacological intervention against multidrug-resistant tuberculosis.

The principles of RNA structure architecture.

Being informational, enzymatic, as well as a nanoscale molecular machine, ribonucleic acid (RNA) permeates all areas of biology and has been exploited in biotechnology as drug and sensor. Here we describe the composition and fundamental properties of RNA and how the single-stranded RNA chains fold and shape certain motifs that are repeatedly observed in different structures. Small and large molecular mass RNA binders are being touched upon, as is the technology for selecting RNA molecules in vitro that bind almost any kind of natural or artificial target. Recognizing the versatility of RNA is expected to foster the development of tools which monitor RNA in the environment, including plants, animals, and patients. Many of the noncoding RNAs are yet to be identified in the rapidly emerging genomes and assigned to functions. It is hoped that these and similar worthwhile goals will be achieved by integrating the efforts of bench and computer scientists.

A novel histone H4 arginine 3 methylation-sensitive histone H4 binding activity and transcriptional regulatory function for signal recognition particle subunits SRP68 and SRP72.

BACKGROUND: Histone methylation is believed to recruit specific histone-binding proteins. RESULTS: We identified SRP68/72 heterodimers as major nuclear proteins whose binding of histone H4 tail is inhibited by H4R3 methylation. CONCLUSION: SRP68/72 are novel histone H4-binding proteins. SIGNIFICANCE: Uncovers a novel chromatin regulatory function for SRP68/72 and suggests that histone arginine methylation may function mainly in inhibiting rather than recruiting effector proteins. Arginine methylation broadly occurs in the tails of core histones. However, the mechanisms by which histone arginine methylation regulates transcription remain poorly understood. In this study we attempted to identify nuclear proteins that specifically recognize methylated arginine 3 in the histone H4 (H4R3) tail using an unbiased proteomic approach. No major nuclear protein was observed to specifically bind to methylated H4R3 peptides. However, H4R3 methylation markedly inhibited the binding of two proteins to H4 tail peptide. These proteins were identified as the SRP68 and SRP72 heterodimers (SRP68/72), the components of the signal recognition particle (SRP). Only SRP68/72, but not the SRP complex, bound the H4 tail peptide. SRP68 and SRP72 bound the H4 tail in vitro and associated with chromatin in vivo. The chromatin association of SRP68 and SRP72 was regulated by PRMT5 and PRMT1. Both SRP68 and SRP72 activated transcription when tethered to a reporter via a heterologous DNA binding domain. Analysis of the genome-wide occupancy of SRP68 identified target genes regulated by SRP68. Taken together, these results demonstrate a role of H4R3 methylation in blocking the binding of effectors to chromatin and reveal a novel role for the SRP68/SRP72 heterodimer in the binding of chromatin and transcriptional regulation.

Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5' leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure-function relationships of this multifaceted primordial ribozyme.

RNAcentral: A vision for an international database of RNA sequences.

During the last decade there has been a great increase in the number of noncoding RNA genes identified, including new classes such as microRNAs and piRNAs. There is also a large growth in the amount of experimental characterization of these RNA components. Despite this growth in information, it is still difficult for researchers to access RNA data, because key data resources for noncoding RNAs have not yet been created. The most pressing omission is the lack of a comprehensive RNA sequence database, much like UniProt, which provides a comprehensive set of protein knowledge. In this article we propose the creation of a new open public resource that we term RNAcentral, which will contain a comprehensive collection of RNA sequences and fill an important gap in the provision of biomedical databases. We envision RNA researchers from all over the world joining a federated RNAcentral network, contributing specialized knowledge and databases. RNAcentral would centralize key data that are currently held across a variety of databases, allowing researchers instant access to a single, unified resource. This resource would facilitate the next generation of RNA research and help drive further discoveries, including those that improve food production and human and animal health. We encourage additional RNA database resources and research groups to join this effort. We aim to obtain international network funding to further this endeavor.

In silico analysis of IRES RNAs of foot-and-mouth disease virus and related picornaviruses.

Foot-and-mouth disease virus (FMDV) uses an internal ribosome entry site (IRES), a highly structured segment of its genomic RNA, to hijack the translational apparatus of an infected host. Computational analysis of 162 type II picornavirus IRES RNA sequences yielded secondary structures that included only base pairs supported by comparative or experimental evidence. The deduced helical sections provided the foundation for a hypothetical three-dimensional model of FMDV IRES RNA. The model was further constrained by incorporation of data derived from chemical modification and enzymatic probing of IRES RNAs as well as high-resolution information about IRES RNA-bound proteins.

Comparative structural studies of bovine viral diarrhea virus IRES RNA.

The internal ribosomal entry site (IRES) RNA of bovine viral diarrhea virus (BVDV) has been implicated in virus propagation. To gain insight into the structure and potential function of the BVDV IRES RNA, we collected and aligned 663 of its sequences. Compensatory Watson-Crick and wobble G·U pairs were investigated to establish phylogenetically supported secondary structures for each of the BVDV IRES RNA sequences. The extensively folded BVDV IRES RNAs were composed of helices 2, 3 and 4. Helix 2 consisted of five helical sections. Helix 3 contained sections 3a to 3j as well as six helical insertions 3.1-3.6. Sections 3a and 3b together with helices 3.6 and 4 formed an RNA pseudoknot. Two highly variable regions corresponded to hairpins 3j and 3.4. Three-dimensional modeling of the BVDV-1b strain Osloss IRES RNA predicted an elongated structure with approximate dimensions of 170 Å by 65 Å by 90 Å. The model of the IRES RNA-ribosome complex suggested proximity between helix 2 of the BVDV IRES and ribosomal proteins S5 and S25.

Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA.

BACKGROUND: Human cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain. RESULTS: We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA. CONCLUSIONS: The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.

Visualizing the transfer-messenger RNA as the ribosome resumes translation.

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.

Archaea signal recognition particle shows the way.

Archaea SRP is composed of an SRP RNA molecule and two bound proteins named SRP19 and SRP54. Regulated by the binding and hydrolysis of guanosine triphosphates, the RNA-bound SRP54 protein transiently associates not only with the hydrophobic signal sequence as it emerges from the ribosomal exit tunnel, but also interacts with the membrane-associated SRP receptor (FtsY). Comparative analyses of the archaea genomes and their SRP component sequences, combined with structural and biochemical data, support a prominent role of the SRP RNA in the assembly and function of the archaea SRP. The 5e motif, which in eukaryotes binds a 72 kilodalton protein, is preserved in most archaea SRP RNAs despite the lack of an archaea SRP72 homolog. The primary function of the 5e region may be to serve as a hinge, strategically positioned between the small and large SRP domain, allowing the elongated SRP to bind simultaneously to distant ribosomal sites. SRP19, required in eukaryotes for initiating SRP assembly, appears to play a subordinate role in the archaea SRP or may be defunct. The N-terminal A region and a novel C-terminal R region of the archaea SRP receptor (FtsY) are strikingly diverse or absent even among the members of a taxonomic subgroup.

Kinship in the SRP RNA family.

The signal recognition particle (SRP) is a ribonucleoprotein complex which participates in the targeting of protein to cellular membranes. The RNA component of the SRP has been found in all domains of life, but the size of the molecule and the number of RNA secondary structure elements vary considerably between the different phylogenetic groups. We continued our efforts to identify new SRP RNAs, compare their sequences, discover new secondary structure elements, conserved motifs, and other properties. We found additional proof for the variability in the apical loop of helix 8, and we identified several bacteria which lack all of their SRP components. Based on the distribution of SRP RNA features within the taxonomy, we suggest seven alignment groups: Bacteria with a small (4.5S) SRP RNA, Bacteria with a large (6S) SRP RNA, Archaea, Fungi (Ascomycota), Metazoa group, Protozoa group, and Plants. The proposed divisions improve the prediction of more distantly related SRP RNAs and provide a more inclusive representation of the SRP RNA family. Updates of the Rfam SRP RNA sequence collection are expected to benefit from the suggested groupings.

Characterization of the SRP68/72 interface of human signal recognition particle by systematic site-directed mutagenesis.

The signal recognition particle (SRP) is a ribonucleoprotein complex which is crucial for the delivery of proteins to cellular membranes. Among the six proteins of the eukaryotic SRP, the two largest, SRP68 and SRP72, form a stable SRP68/72 heterodimer of unknown structure which is required for SRP function. Fragments 68e' (residues 530 to 620) and 72b' (residues 1 to 166) participate in the SRP68/72 interface. Both polypeptides were expressed in Escherichia coli and assembled into a complex which was stable at high ionic strength. Disruption of 68e'/72b' and SRP68/72 was achieved by denaturation using moderate concentrations of urea. The four predicted tetratricopeptide repeats (TPR1 to TPR4) of 72b' were required for stable binding of 68e'. Site-directed mutagenesis suggested that they provide the structural framework for the binding of SRP68. Deleting the region between TPR3 and TPR4 (h120) also prevented the formation of a heterodimer, but this predicted alpha-helical region appeared to engage several of its amino acid residues directly at the interface with 68e'. A 39-residue polypeptide (68h, residues 570-605), rich in prolines and containing an invariant aspartic residue at position 585, was found to be active. Mutagenesis scanning of the central region of 68h demonstrated that D585 was solely responsible for the formation of the heterodimer. Coexpression experiments suggested that 72b' protects 68h from proteolytic digestion consistent with the assertion that 68h is accommodated inside a groove formed by the superhelically arranged four TPRs of the N-terminal region of SRP72.

Variations on the tmRNA gene.

tmRNA employs both tRNA-like and mRNA-like properties as it rescues stalled bacterial ribosomes, while targeting the defective mRNA and incomplete nascent protein for degradation. We describe variation of the tmRNA gene (ssrA) and how it informs tmRNA structure and function. Endosymbiont tmRNAs tend to lose secondary structure and length in the mRNA-like region as nucleotide composition drifts with that of the whole genome. A dramatic gene structure variation is circular permutation, which produces two-piece tmRNAs in three bacterial lineages; new sequences blur these lineages. We present evidence that Sinorhizobium two-piece tmRNA retains the 5'-triphosphate of transcriptional initiation and predict a new structure at the 5' end of cyanobacterial two-piece tmRNA precursor. ssrA is a target for some mobile DNAs and a passenger on others. It has been found interrupted (but not functionally disrupted) by mobile elements such as group I introns, genomic islands and palindromic elements. The alphaproteobacterial permuted genes are significantly less frequently interrupted by genomic islands than are their standard counterparts, yet are a hotspot for insertion or swapping of rickettsial palindromic elements, in contrast to other rickettsial loci that show steady decay of a single ancestral element. Bacteriophages, plasmids and genomic islands can carry tmRNA genes; we describe a native bacterial ssrA disrupted by insertion of a genomic island that carries its own ssrA, a genome encoding both one- and two-piece tmRNA, and a phage encoding a tmRNA variant lacking the mRNA-like function, which may counteract host tmRNA during infection.