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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Binding of SRSF4 to a novel enhancer modulates splicing of exon 6 of Fas pre-mRNA

Highlights: • SRSF4 expression promotes exon 6 inclusion of Fas pre-mRNA. • A stronger 5′ splice-site of exon 6 is required for SRSF4-mediated regulation on exon 6 splicing. • SRSF4 functionally targets GGGGUG RNA sequence on exon 6. • SRSF4 directly interacts with GGGGUG sequence on exon 6. Alternative splicing of exon 6 in Fas pre-mRNA generates a membrane bound pro-apoptotic isoform or soluble anti-apoptotic isoform. SRSF4 is a member of Arginine-Serine rich (SR) protein family. Here we demonstrate that increased SRSF4 expression stimulates exon 6 inclusion, and that reduced SRSF4 expression promotes exon 6 exclusion. We also show that weaker but not stronger 5’ splice-site strength of exon 6 abolishes the SRSF4 effects on exon 6 splicing. Furthermore, we identified a novel enhancer on exon 6, on which SRSF4 interacts functionally and physically. Our results illustrate a novel regulatory mechanism of Fas pre-mRNA splicing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN–nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Tissue-specific variation in 5-terminal exons of mouse Anoctamin 1 transcript induces N-terminal variation of its protein via alternative translational start sites

Anoctamin 1 (encoded by the Ano1 gene) is a Ca{sup 2+}-activated Cl{sup −} channel critical to many physiological functions. It has been speculated that Ano1 expression is regulated in a tissue-dependent manner via alternative promoters. However, variation in the 5-end sequence of mouse Ano1 (mAno1) and its tissue-dependent regulation are poorly understood. We identified a novel 5-terminal exon (designated exon 1a) of mAno1 instead of the known 5-terminal exon (exon 0) using 5-rapid amplification of cDNA ends (RACE) analysis. Unexpectedly, the novel 5-end variant mAno1{sub Ex1a} was abundantly expressed in many tissues including the salivary and mammary glands, rectum, lung, trachea and prostate. In contrast, the known variant mAno1{sub Ex0} predominated only in male reproductive tissues such as the epididymis and testis. In a heterologous expression system, mAno1{sub Ex0} encoded a longer protein than mAno1{sub Ex1a}, and this long isoform was abolished by a mutation in the exon 0 start codon. Moreover, the mAno1{sub Ex0}-specific N-terminal sequence was immunohistochemically detected in epididymis but not in salivary gland. Our data suggest that mAno1 expression is regulated via alternative promoters, and its transcriptional variation results in variation of the N-terminal sequence of the Ano1 protein due to the alternative translation initiation sites. These tissue-specific variations might contribute to the regulation of mAno1 expression and activity according to the physiological function of each tissue.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ~80% to ~20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Splicing dysregulation contributes to the pathogenicity of several F9 exonic point variants

Abstract Background Pre‐mRNA splicing is a complex process requiring the identification of donor site, acceptor site, and branch point site with an adjacent polypyrimidine tract sequence. Splicing is regulated by splicing regulatory elements (SREs) with both enhancer and suppressor functions. Variants located in exonic regions can impact splicing through dysregulation of native splice sites, SREs, and cryptic splice site activation. While splicing dysregulation is considered primary disease‐inducing mechanism of synonymous variants, its contribution toward disease phenotype of non‐synonymous variants is underappreciated. Methods In this study, we analyzed 415 disease‐causing and 120 neutral F9 exonic point variants including both synonymous and non‐synonymous for their effect on splicing using a series of in silico splice site prediction tools, SRE prediction tools, and in vitro minigene assays. Results The use of splice site and SRE prediction tools in tandem provided better prediction but were not always in agreement with the minigene assays. The net effect of splicing dysregulation caused by variants was context dependent. Minigene assays revealed that perturbed splicing can be found. Conclusion Synonymous variants primarily cause disease phenotype via splicing dysregulation while additional mechanisms such as translation rate also play an important role. Splicing dysregulation is likely to contribute to the disease phenotype of several non‐synonymous variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Huntingtin structure is orchestrated by HAP40 and shows a polyglutamine expansion-specific interaction with exon 1

Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. Here, we demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease and illuminate the structural consequences of HTT polyglutamine expansion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Hemiptera phylogenomic resources: Tree‐based orthology prediction and conserved exon identification

Abstract High‐throughput sequencing of transcriptomes and targeted genomic regions are advancing our knowledge of The Tree of Life. Building phylogenies with regions of the genome requires 1‐to‐1 orthologue resources of genes and noncoding loci. One organismal group that has received little attention in this area is the Hemiptera, the fifth largest insect order represented by ~103,590 named species. Here, we present a set of 3,872 Hemiptera 1‐to‐1 orthogroups based on tree‐based orthology inference of eight Hemiptera species with publicly available genome sequences. We also estimate a set of 406 orthologous exons with similar mRNA splice sites that can be used for Sanger sequencing and develop enrichment probes for targeted genome sequencing for phylogenomic inference. We show this novel set of orthologues is informative at the protein, coding sequence and exon molecular levels and provides robust branch support in both gene tree–species tree methods and concatenated sequence phylogenies. In addition, we demonstrate the utility of these loci to resolve relationships in whiteflies, Bemisia tabaci , a large species complex with few phylogenomic resources. Last, we compare our Hemiptera phylogeny with previously published phylogenies and other orthologue databases, while providing suggestions on further improvement to this phylogenomic resource.

Owen, Christopher L.↗
DOE OSTI2021

A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Exon disruptive variants in Populus trichocarpa associated with wood properties exhibit distinct gene expression patterns

Abstract Forest trees may harbor naturally occurring exon disruptive variants (DVs) in their gene sequences, which potentially impact important ecological and economic phenotypic traits. However, the abundance and molecular regulation of these variants remain largely unexplored. Here, 24,420 DVs were identified by screening 1014Populus trichocarpafull genomes. The identified DVs were predominantly heterozygous with allelic frequencies below 5% (only 26% of DVs had frequencies greater than 5%). Using common garden‐grown trees, DVs were assessed for gene expression variation in the developing xylem, revealing that their gene expression can be significantly altered, particularly for homozygous DVs (in the range of 27%–38% of cases depending on the studied common garden). DVs were further investigated for their correlations with 13 wood quality traits, revealing that, among the 148 discovered DV associations, 15 correlated with more than one wood property and six genes had more than one DV in their coding sequences associated with wood traits. Approximately one‐third of DVs correlated with wood property variation also showed significant gene expression variation, confirming their non‐spurious impact. These findings offer potential avenues for targeted introduction of homozygous mutations using tree biotechnology, and while the exact mechanisms by which DVs may directly influence wood formation remain to be unraveled, this study lays the groundwork for further investigation.

Genetics & Heredity↗
DOE OSTI2021

Cryo-electron tomography provides topological insights into mutant huntingtin exon 1 and polyQ aggregates

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Circ-UBR5: An exonic circular RNA and novel small nuclear RNA involved in RNA splicing

Highlights: • Low expression of circ-UBR5 in NSCLC was significantly related with tumor poor differentiation. • A highly efficient strategy for finding specific circRNA binding proteins using HuprotTM Protoarray was first be presented. • Circ-UBR5 could bind splicing regulatory factors QKI, and NOVA1 and U1 snRNA in the nucleus. • Our research first revealed the potential function of a non-functional phenotype circRNA in NSCLC cells. Circular RNAs (circRNAs) are class of non-coding RNAs formed by back-splicing events as loops, and could be found in all types of organisms. They play important and diverse roles in cell development, growth, and tumorigenesis, but functions of the majority of circRNAs remain enigmatic. Particularly functional phenotypes of great majority of circRNAs are not obvious. Here we randomly selected a circRNA circ-UBR5, which has no obvious functional phenotype in non-small cell lung cancer (NSCLC) cells from our previous research findings, to explore its potential function in cells. Differential expression of circ-UBR5 was detected in paired samples of tumorous tissues and adjacent nontumorous tissues from 59 patients with NSCLC by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Results showed circ-UBR5 expression was significantly downregulated in NSCLC tissues (p (p = 0.00126), suggesting circ-UBR5 might serve as an index of NSCLC differentiation. Our findings indicated circ-UBR5 could bind splicing regulatory factor QKI, KH domain containing RNA binding (QKI) and NOVA alternative splicing regulator 1 (NOVA1) and U1 small nuclear RNA (snRNA) in the nucleus, revealing circ-UBR5 might be a novel snRNA involved in RNA splicing regulatory process. Moreover, we first presented a highly efficient strategy for finding specific circRNA binding proteins using Human Protein Microarray (Huprot™ Protoarray).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR.

59 BASIC BIOLOGICAL SCIENCES↗