Non-canonical Elements Research Articles

Abstract 5862: Retrotransposable elements mediate the drug-tolerant persistence in claudin-low breast cancer chemo-treatment

Abstract The emergence of acquired drug resistance through treatment remains a critical threat to efficient cancer therapies, which most often lead to relapse and metastasis. The development of resistance is an evolutionary adaptation process that heavily depends on tumor heterogeneity (underpinned by cellular plasticity). Therefore, a longitudinal deconvolution of treatment adaptation is critical to uncover the driving-force mechanisms underpinning resistance adaptation. In the clinic, chemotherapy remains the mainstream treatment for TNBC, and one of the primary chemo-agents is doxorubicin. Although the initial responsive rate of doxorubicin-based chemotherapies is up to 70%, it is well recognized that TNBC cells usually generate an adaptive response and result in acquired drug-resistance and multi-drug resistant phenotypes. Although numerous mechanisms of chemo-resistance evolution have been proposed, most of these studies focused on the initial and terminal states, from which we could not deconvolute the adaptation routes and might miss the transient fate-switching events. We hypothesize that the claudin-low TNBC chemo-resistant cells may derive from the early-stage reversible chemo-tolerant persistent state, and early-stage state-switching mediated by epigenetic landscape reprogramming might determine the adaptation through treatment. We developed an in vitro “pulsing-treatment” model to test the hypothesis, which could mimic the clinical treatment and provide therapeutically relevant insights into the acute drug-induced stress response and regrowth. Unexpectedly, we found that the human endogenous retrovirus-like elements (HERVs) show an aberrant bursting in the adaptation. To further interrogate the adaptation, we did the longitudinal single-cell multi-omics sequencing. With a novel bioinformatic workflow, we integrated the HERV’s expression information with protein-coding genes profiling and chromatin accessibility. The preliminary result indicated that a subpopulation of HERV-high cells might transiently mediate the persistence of cancer cells at the early stage of the treatment, which coupled with whole transcriptomic and chromatin landscape reprogramming, and molecular subtype switching. In this study, we employed a new strategy to investigate the longitudinal adaptation route through treatment and uncover a non-canonical element, which shed new light on drug resistance research and novel target screening. Citation Format: Zijian Zhang, Yiyang Wang, Xinlong Luo, Xiaomei Zhan, Jun Ding, Tao P. Wu. Retrotransposable elements mediate the drug-tolerant persistence in claudin-low breast cancer chemo-treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5862.

Structural analysis of the Trypanosoma brucei EIF4E6/EIF4G5 complex reveals details of the interaction between unusual eIF4F subunits

Recognition of the mRNA 5′ end is a critical step needed for translation initiation. This step is performed by the cap binding protein eIF4E, which joins the larger eIF4G subunit to form the eIF4F complex. Trypanosomatids have a minimum of five different eIF4F-like complexes formed through specific but not well-defined interactions between four different eIF4E and five eIF4G homologues. The EIF4E6/EIF4G5 complex has been linked with the stage-specific translation of mRNAs encoding the major Trypanosoma brucei virulence factors. Here, to better define the molecular basis for the TbEIF4E6/TbEIF4G5 interaction, we describe the identification of the peptide interacting with TbEIF4E6 in the region comprising residues 79–166 of TbEIF4G5. The TbEIF4E6-TbEIF4G5_79-116 complex reconstituted with recombinant proteins is highly stable even in the absence of cap-4. The crystal structure of the complex was subsequently solved, revealing extensive interacting surfaces. Comparative analyses highlight the conservation of the overall structural arrangement of different eIF4E/eIF4G complexes. However, highly different interacting surfaces are formed with distinct binding contacts occurring both in the canonical and noncanonical elements within eIF4G and the respective eIF4E counterpart. These specific pairs of complementary interacting surfaces are likely responsible for the selective association needed for the formation of distinct eIF4F complexes in trypanosomatids.

High-resolution structure of stem-loop 4 from the 5'-UTR of SARS-CoV-2 solved by solution state NMR.

We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonancespectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a G•U wobble base pair and a pyrimidine•pyrimidine mismatch, which are incorporated into the stem-loop structure. Boththe tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.

A vast evolutionarily transient translatome contributes to phenotype and fitness.

Translation is the process by which ribosomes synthesize proteins. Ribosome profiling recently revealed that many short sequences previously thought to be noncoding are pervasively translated. To identify protein-coding genes in this noncanonical translatome, we combine an integrative framework for extremely sensitive ribosome profiling analysis, iRibo, with high-powered selection inferences tailored for short sequences. We construct a reference translatome for Saccharomyces cerevisiae comprising 5,400 canonical and almost 19,000 noncanonical translated elements. Only 14 noncanonical elements were evolving under detectable purifying selection. A representative subset of translated elements lacking signatures of selection demonstrated involvement in processes including DNA repair, stress response, and post-transcriptional regulation. Our results suggest that most translated elements are not conserved protein-coding genes and contribute to genotype-phenotype relationships through fast-evolving molecular mechanisms.

The Genomic Response to TGF-β1 Dictates Failed Repair and Progression of Fibrotic Disease in the Obstructed Kidney.

Tubulointerstitial fibrosis is a common and diagnostic hallmark of a spectrum of chronic renal disorders. While the etiology varies as to the causative nature of the underlying pathology, persistent TGF-β1 signaling drives the relentless progression of renal fibrotic disease. TGF-β1 orchestrates the multifaceted program of kidney fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery or re-differentiation, capillary collapse and subsequent interstitial fibrosis eventually leading to chronic and ultimately end-stage disease. An increasing complement of non-canonical elements function as co-factors in TGF-β1 signaling. p53 is a particularly prominent transcriptional co-regulator of several TGF-β1 fibrotic-response genes by complexing with TGF-β1 receptor-activated SMADs. This cooperative p53/TGF-β1 genomic cluster includes genes involved in cellular proliferative control, survival, apoptosis, senescence, and ECM remodeling. While the molecular basis for this co-dependency remains to be determined, a subset of TGF-β1-regulated genes possess both p53- and SMAD-binding motifs. Increases in p53 expression and phosphorylation, moreover, are evident in various forms of renal injury as well as kidney allograft rejection. Targeted reduction of p53 levels by pharmacologic and genetic approaches attenuates expression of the involved genes and mitigates the fibrotic response confirming a key role for p53 in renal disorders. This review focuses on mechanisms underlying TGF-β1-induced renal fibrosis largely in the context of ureteral obstruction, which mimics the pathophysiology of pediatric unilateral ureteropelvic junction obstruction, and the role of p53 as a transcriptional regulator within the TGF-β1 repertoire of fibrosis-promoting genes.

The biomedical challenge of neurodegenerative disorders: an opportunity for cannabinoid-based therapies to improve on the poor current therapeutic outcomes.

At the beginning of the 21st century, the therapeutic management of neurodegenerative disorders remains a major biomedical challenge, particularly given the worldwide ageing of the population over the past 50years that is expected to continue in the forthcoming years. This review will focus on the promise of cannabinoid-based therapies to address this challenge. This promise is based on the broad neuroprotective profile of cannabinoids, which may cooperate to combat excitotoxicity, oxidative stress, glia-driven inflammation and protein aggregation. Such effects may be produced by the activity of cannabinoids through their canonical targets (e.g. cannabinoid receptors and endocannabinoid enzymes) and also via non-canonical elements and activities in distinct cell types critical for cell survival or neuronal replacement (e.g. neurons, glia and neural precursor cells). Ultimately, the therapeutic events driven by endocannabinoid signalling reflect the activity of an endogenous system that regulates the preservation, rescue, repair and replacement of neurons and glia. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Kinetics and Thermodynamics of Non-Canonical DNA

Many native nucleic acid hairpins contain various non-canonical structures, including mismatches, bubbles and loops. The thermodynamic landscape of these hairpins containing these motifs are not well understood. We combine optical tweezers with an energy based model to reconstruct the energy landscape for DNA hairpin unfolding. Overall hairpin stability, and the unfolding kinetics show the destabilizing effects of non-canonical elements in DNA hairpin structure. Finally, the stabilizing effect of the rhodium coordination complex [Rh(bpy)2(chrysi)]3+ is probed. Rhodium chrysi preferentially binds to defects and mismatches in hairpin stems, enhancing duplex stability. Rhodium chrysi binding thus allows interrupted hairpins to resemble fully formed ones.

Distinct brain signatures of content and structure violation during action observation

Sentences, musical phrases and goal-directed actions are composed of elements that are linked by specific rules to form meaningful outcomes. In goal-directed actions including a non-canonical element or scrambling the order of the elements alters the action’s content and structure, respectively. In the present study we investigated event-related potentials of the electroencephalographic (EEG) activity recorded during observation of both alterations of the action content (obtained by violating the semantic components of an action, e.g. making coffee with cola) and alterations of the action structure (obtained by inverting the order of two temporally adjacent pictures of sequences depicting daily life actions) interfering with the normal flow of the motor acts that compose an action. Action content alterations elicited a bilateral posterior distributed EEG negativity, peaking at around 400ms after stimulus onset similar to the ERPs evoked by semantic violations in language studies. Alteration of the action structure elicited an early left anterior negativity followed by a late left anterior positivity, which closely resembles the ERP pattern found in language syntax violation studies. Our results suggest a functional dissociation between the processing of action content and structure, reminiscent of a similar dissociation found in the language or music domains. Importantly, this study provides further support to the hypothesis that some basic mechanisms, such as the rule-based structuring of sequential events, are shared between different cognitive domains.

Preverbal subjects in Galician: Experimental data in the A vs. A′ debate

The debate over whether preverbal subjects of transitive verbs are canonical or non-canonical elements in Iberian Romance and other null-subject languages over the past twenty-plus years has arguably left the field of Generative syntax at an impasse with respect to the issue. Researchers have argued quite convincingly that preverbal subjects are non-canonical, non-argument (A′) constituents (e.g. Barbosa 1996, 2000; Ordóñez and Treviño 1999; Uribe-Etxebarria 1992, 1995), while others have presented equally compelling evidence supporting the analysis of preverbal subjects as canonical argument (A) constituents (e.g. Costa 2004; Goodall 2001). The results of this debate are of great theoretical importance, as they been claimed to have implications for the universality of the EPP and its associated feature(s). In this paper, I present evidence for Galician in the spirit of previous research and show that, with respect to the standard syntactic tests of A-versus A′-status for preverbal subjects in the literature, Galician is also inconclusive. I discuss how an experimental methodology incorporating a structured discourse context may provide greater insight on the issue and, assuming López's (2009) analysis of CLLD elements, present evidence suggesting that preverbal subjects are not left-peripheral, A′-elements in the discourse contexts examined in Galician, but rather A-elements.

Intronic DNA Elements Regulate Androgen-Dependent Expression of the Murine Nkx3.1 Gene

Nkx3.1 is a well-conserved homeobox gene that is involved in development, differentiation and maintenance of prostate epithelial cells. Nkx3.1 expression is induced by androgen in prostate epithelia and, as such, our interest is to understand the mechanism(s) for this androgen-dependent expression in normal epithelial cells. In this report, we show that the region of DNA sequence 2.7 kilobases in front of the mouse Nkx3.1 gene drives enhanced transcription in prostate epithelia cells; however, this segment was not capable of androgen-directed regulation. Among the multiple, potential androgen response elements (AREs) identified by scanning sequences near and within the gene, two sequences within the intron of the murine Nkx3.1 gene were demonstrated to confer androgen-dependent transcription in reporter gene transfection experiments. Each of the elements, termed ARE A and ARE B, contained a 6-base pair core sequence, TGTTCT, that has been described as an androgen receptor half-site binding sequence, separated by 498 base pairs of DNA. Both of the intronic half-sites bind activated androgen receptor from a variety of sources, albeit with different apparent affinities. This region of the Nkx3.1 gene demonstrates a high degree of conservation among diverse species and mutagenesis experiments demonstrated that both elements are required for androgen stimulation. Taken together, our study shows that androgen-dependent transcription of the mouse Nkx3.1 gene is conferred through a noncanonical element within the intron of the gene.

Symmetries of Ginsparg-Wilson chiral fermions

The group structure of the variant chiral symmetry discovered by Luescher in the Ginsparg-Wilson description of lattice chiral fermions is analyzed. It is shown that the group contains an infinite number of linearly independent symmetry generators, and the Lie algebra is given explicitly. CP is an automorphism of this extended chiral group, and the CP transformation properties of the symmetry generators are found. The group has an infinite-parameter invariant subgroup, and the factor group, whose elements are its cosets, is isomorphic to the continuum chiral symmetry group. Features of the currents associated with these symmetries are discussed, including the fact that some different, noncommuting symmetry generators lead to the same Noether current. These are universal features of lattice chiral fermions based on the Ginsparg-Wilson relation; they occur in the overlap, domain-wall, and perfect-action formulations. In a solvable example, free overlap fermions, these noncanonical elements of lattice chiral symmetry are related to complex energy singularities that violate reflection positivity and impede continuation to Minkowski space.

Crystal Structure of the Pml1p Subunit of the Yeast Precursor mRNA Retention and Splicing Complex

The precursor mRNA retention and splicing (RES) complex mediates nuclear retention and enhances splicing of precursor mRNAs. The RES complex from yeast comprises three proteins, Snu17p, Bud13p and Pml1p. Snu17p acts as a central platform that concomitantly binds the Bud13p and Pml1p subunits via short peptide epitopes. As a step to decipher the molecular architecture of the RES complex, we have determined crystal structures of full-length Pml1p and N-terminally truncated Pml1p. The first 50 residues of full-length Pml1p, encompassing the Snu17p-binding region, are disordered, showing that Pml1p binds to Snu17p via an intrinsically unstructured region. The remainder of Pml1p folds as a forkhead-associated (FHA) domain, which is expanded by a number of noncanonical elements compared with known FHA domains from other proteins. An atypical N-terminal appendix runs across one β-sheet and thereby stabilizes the domain as shown by deletion experiments. FHA domains are thought to constitute phosphopeptide-binding elements. Consistently, a sulfate ion was found at the putative phosphopeptide-binding loops of full-length Pml1p. The N-terminally truncated version of the protein lacked a similar phosphopeptide mimic but retained an almost identical structure. A long loop neighboring the putative phosphopeptide-binding site was disordered in both structures. Comparison with other FHA domain proteins suggests that this loop adopts a defined conformation upon ligand binding and thereby confers ligand specificity. Our results show that in the RES complex, an FHA domain of Pml1p is flexibly tethered via an unstructured N-terminal region to Snu17p.

Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network.

American Incunabula: 'Grotesque Genesis' and the Genealogical Genre

Analysis of Os Sertões (Euclides da Cunha, Brazil, 1902), Absalom, Absalom! (William Faulkner, USA, 1936), Cien años de soledad (Gabriel García Márquez, Colombia, 1967), The Invention of the World (Jack Hodgins, Canada, 1977), and Texaco (Patrick Chamoiseau, Martinique, 1992) as a generic ensemble enables diverse treatments of race, class, gender and sexuality to resolve over time and across cultures into the meaningful patterns of American literary history. Each text incorporates the origin in writing and exposes it to difference—plurality, ambiguity, discontinuity. With this, the perpetual rewriting of the strong poem (the Book of Genesis) at the symbolic founding, the originary tradition transforms itself through incorporation of non-canonical elements, as the ‘same’ turns endlessly different: hybrid, ex-centric, grotesque, increasingly Creolized.

Extending partial pro-drop in Modern Hebrew: A comprehensive analysis

Modern Hebrew is considered to be a 'partial pro-drop language'. Traditionally, the distinction between cases where pro-drop is licensed and those in which it is prohibited, was based on the person and tense features of the verb: 1st and 2nd person pronominal subjects may be omitted in past and future tense. This generalization, however, was found to be false in a number of papers, each discussing a subset of the data. Thus, contrary to conventional wisdom, dropped 3rd person pronouns subjects do occur in the language in particular contexts. Identifying these contexts by way of a corpus-based survey is the initial step taken in this study. Subsequently, a careful syntactic analysis of the data reveals broad generalizations which have not been made to date. Thus, what was initially assumed to be a uniform phenomenon of 3rd person pro-drop turns out to be manifested in three distinct types of constructions. Finally, the proposed HPSG-based analysis incorporates insights concerning locality, correlations between finite and non-finite control, non-canonical elements, and binding.

Non-optimal TATA Elements Exhibit Diverse Mechanistic Consequences

To reveal mechanistic differences in transcription initiation between variant TATA elements, in vivo and in vitro assays of the functional activity of 14 different sequences were compared. Variant elements exhibited particular degrees of activation in vivo but universally were unable to support the -fold activation observed for an element consisting of TATAAA. Each element was classified by its functional activity for in vitro interaction with TATA-binding protein (TBP), TFIIA, and TFIIB. Certain off-consensus TATA elements form poor binding sites for TBP and this compromised interaction interferes with higher order complex formation with TFIIA and/or TFIIB. Other elements are only modestly decreased for TBP binding but dramatically affected for higher order complex formation. Another distinct category is comprised of two elements (CATAAA and TATAAG), which are not affected in the initial formation of the TBP, TFIIA-TBP, or TFIIB-TBP complexes. However, CATAAA and TATAAG are unable to form a stable TFIIA-TBP-DNA complex in vitro. Moreover, fusion of TFIIA to TBP specifically restores activity from these two elements in vivo. Taken together, these results indicate that the interplay between the sequence of the TATA element and the components of the general transcription machinery can lead to variations in the formation of functional complexes and/or the stability of these complexes. These differences offer distinct opportunities for an organism to exploit diverse steps in the regulation of gene expression depending on the precise TATA element sequence at a given gene.

Deletion in a (T)8 microsatellite abrogates expression regulation by 3'-UTR.

A high level of genetic instability might cause mutations to accumulate in tumours. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. By scanning transcriptome databases, we found that microsatellites in the human genome are less numerous in coding DNA than in the 3'-untranslated region (UTR), known to mediate control of gene expression. By mutation analysis, we identified a 1 bp deletion in a (T)(8) microsatellite embedded in the 1801 nucleotide long 3'-UTR of CEACAM1 gene, thought to be involved in tumour onset and progression. By Lentiviral Vector- mediated gene transfer, we showed that the wild-type but not the mutated CEACAM1 3'-UTR greatly decreased transgene expression at both mRNA and protein level. Messenger RNA abundance was fully regulated by the most 3' region of CEACAM1 3'-UTR. This region includes the (T)(8) microsatellite but not any known classified regulatory element. These data show that CEACAM1 3'-UTR contains non-canonical elements contributing to mRNA regulation, among which a short repeat sequence could play a critical regulatory function. This suggests that, in cancer cells, a single mutation in a 3'-UTR short microsatellite might strongly affect gene expression.

Structural Heterogeneity and Genomic Distribution of Drosophila melanogaster LTR-Retrotransposons

Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, beta-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.

RNA recognition by arginine-rich peptide motifs.

A ubiquitious class of RNA-binding proteins is distinguished by an arginine-rich motif. Such proteins function in transcription, translation, RNA trafficking, and packaging. Peptide models are derived from viral regulatory proteins, including the virulence factors Tat and Rev of mammalian immunodeficiency viruses. Structures of model peptide-RNA complexes exhibit diverse strategies of recognition based in each case on structural transitions. Induced RNA structures contain noncanonical elements such as purine-purine mismatches, base triples, and flipped bases. Such elements enlarge and extend the RNA major groove to create specific peptide-binding pockets and surfaces. The repertoire of bound peptide structures--beta-hairpin, alpha-helix, and helix-bend-helix-reflects the diversity of induced RNA architectures. This repertoire, reminiscent of primordial exon-encoded peptides, may recapitulate early events in the transition between RNA and protein worlds. Peptide-directed changes in modern RNA structures can provide a mechanism of signaling in higher-order RNA-protein assemblies.

A second-order solution of the main problem of artificial satellitesusing multiple scales

A complete second-order solution of the main problem in the theory of artificial satellites has been developed using the method of multiple scales. The basic equations of motion are expressed in terms of eight nonsingular noncanonical elements with two simple redundancies and an inertial angle as the dependent variable. The obtained solution is expressed in the form of Fourier series whose trigonometric arguments are a linear combination of the unperturbed true anomaly and argument of perigee. The amplitudes of the Fourier series were found to be a nonlinear combination of the unperturbed inclination and eccentricity. The highest frequency of oscillation is found to occur in the eccentricity vector components along the radial and transverse directions of the osculating orbit and is produced by the seventh harmonic in the unperturbed true anomaly.