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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Alternative Splicing Events

Inclusion levels of alternative splicing (AS) events of five different varieties (i.e. skipped exon (SE), retained intron (RI), alternative 5’ splice site (A5SS), alternative 3’ splice site (A3SS), and mutually exclusive exons (MXE)) were measured in human blood samples from two separate cohorts of patients. Cohort 1 (Training Cohort): 12 healthy controls; 12 new onset type 1 diabetic (T1D) cases cases and controls matched on biological sex, age, and body mass index (BMI) 180 million reads Cohort 2 (Testing Cohort): 12 healthy controls; 12 new onset type 1 diabetic (T1D) cases cases and controls matched on biological sex and age. BMI not recorded. 150 million reads

Webb-Robertson, Bobbie-Jo M↗
DOE OSTI2023

Human Islet Research Network (HIRN): Alternative Splicing Events

Inclusion levels of alternative splicing (AS) events of five different varieties (i.e. skipped exon (SE), retained intron (RI), alternative 5’ splice site (A5SS), alternative 3’ splice site (A3SS), and mutually exclusive exons (MXE)) were measured in human blood samples from two separate cohorts of patients. Cohort 1 (Training Cohort): 12 healthy controls; 12 new onset type 1 diabetic (T1D) cases cases and controls matched on biological sex, age, and body mass index (BMI) 180 million reads Cohort 2 (Testing Cohort): 12 healthy controls; 12 new onset type 1 diabetic (T1D) cases cases and controls matched on biological sex and age. BMI not recorded. 150 million reads

Webb-Robertson, Bobbie-Jo M↗
DOE OSTI2020

Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF60 2 :AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Alternative splicing: transcriptional regulatory network in agroforestry

Alternative splicing (AS) in plants plays a key role in regulating the expression of numerous transcripts from a single gene in a regulatory pathway. Variable concentrations of growth regulatory hormones and external stimuli trigger alternative splicing to switch among different growth stages and adapt to environmental stresses. In the AS phenomenon, a spliceosome causes differential transcriptional modifications in messenger RNA (mRNAs), resulting in partial or complete retention of one or more introns as compared to fully spliced mRNA. Differentially expressed proteins translated from intron-retaining messenger RNA (mRNA ir ) perform vital functions in the feedback mechanism. At the post-transcriptional level, AS causes the remodeling of transcription factors (TFs) by the addition or deletion of binding domains to activate and/or repress transcription. In this study, we have summarized the specific role of AS in the regulation of gene expression through repression and activation of the transcriptional regulatory network under external stimuli and switch among developmental stages.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Two Alternative Splicing Variants of AtERF73/HRE1, HRE1α and HRE1β, Have Differential Transactivation Activities in Arabidopsis

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1β. In this study, we examined the differences between the molecular functions of HRE1α and HRE1β. We found that HRE1α and HRE1β are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1β, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1β also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1β was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1β might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1β differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Stress-Induced Changes in Alternative Splicing Landscape in Rice: Functional Significance of Splice Isoforms in Stress Tolerance

Improvements in yield and quality of rice are crucial for global food security. However, global rice production is substantially hindered by various biotic and abiotic stresses. Making further improvements in rice yield is a major challenge to the rice research community, which can be accomplished through developing abiotic stress-resilient rice varieties and engineering durable agrochemical-independent pathogen resistance in high-yielding elite rice varieties. This, in turn, needs increased understanding of the mechanisms by which stresses affect rice growth and development. Alternative splicing (AS), a post-transcriptional gene regulatory mechanism, allows rapid changes in the transcriptome and can generate novel regulatory mechanisms to confer plasticity to plant growth and development. Mounting evidence indicates that AS has a prominent role in regulating rice growth and development under stress conditions. Several regulatory and structural genes and splicing factors of rice undergo different types of stress-induced AS events, and the functional significance of some of them in stress tolerance has been defined. Both rice and its pathogens use this complex regulatory mechanism to devise strategies against each other. This review covers the current understanding and evidence for the involvement of AS in biotic and abiotic stress-responsive genes, and its relevance to rice growth and development. Furthermore, we discuss implications of AS for the virulence of different rice pathogens and highlight the areas of further research and potential future avenues to develop climate-smart and disease-resistant rice varieties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Alternative splicing of OsGS1;1 affects nitrogen‐use efficiency, grain development, and amylose content in rice

SUMMARY Excessive nitrogen fertilizer application is harmful to the environment and reduces the quality of cereal crops. Maintaining crop yields under low nitrogen (LN) conditions and improving quality are important goals for cereal crop breeding. Although the effects of nitrogen assimilation on crop nitrogen‐use efficiency (NUE) have been intensively studied, natural variations of the key assimilation genes underlying grain development and quality are largely unclear. Here, we identified an NUE‐associated gene, OsGS1;1 , encoding glutamine synthase, through genome‐wide association analysis, followed by validation experiments and functional analysis. Fifteen single‐nucleotide polymorphisms in the OsGS1;1 region led to alternative splicing that generated two functional transcripts: OsGS1;1a and OsGS1;1b . The elite haplotype of OsGS1;1 showed high OsGS1;1b activity, which improved NUE, affected grain development, and reduced amylose content. The results show that OsGS1;1 , which is induced under LN conditions, affects grain formation by regulating sugar metabolism and may provide a new avenue for the breeding of high‐yield and high‐quality rice ( Oryza sativa ).

Liu, Xiaolan↗
DOE OSTI2021

U2AF - Hypoxia-induced fas alternative splicing regulator

The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many disease states. Recent studies have revealed that tumorigenesis and hypoxia are involved in large-scale alterations in alternative pre-mRNA splicing. Fas pre-mRNA is alternatively spliced by excluding exon 6 to produce soluble Fas (sFas) protein that lacks a transmembrane domain and acts by inhibiting Fas mediated apoptosis. }In the present study we show that U2AF is involved in hypoxia dependent anti-apoptotic Fas mRNA isoform formation. Our performed studies show that U2AF-RNA interaction is reduced in hypoxic cells, leading to reduction of Fas and increased sFas mRNAs formation. Efficient U2AF-RNA interactions of both subunits are important for Fas exon 6 inclusion into forming mRNA in normoxic and hypoxic cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

transcription start and end sites↗
DOE OSTI2024

AtDGCR14L contributes to salt-stress tolerance via regulating pre-mRNA splicing in Arabidopsis

In plants, the pre-mRNA alternative splicing has been demonstrated to be a crucial tier that regulates gene expression in response to salt stress. However, the underlying mechanisms remain elusive. Here, in this study, we studied the roles of DIGEORGE-SYNDROME CRITICAL REGION 14-like (AtDGCR14L) in regulating pre-mRNA splicing and salt stress tolerance. We discovered that Arabidopsis AtDGCR14L is required for maintaining plant salt stress tolerance and the constitutively spliced and active isoforms of important stress- and/or abscisic acid (ABA)-responsive genes. We also identified the interaction between AtDGCR14L and splicing factor U1-70k, which needs a highly conserved three amino acid (TWG) motif in DGCR14. Different from wild-type AtDGCR14L, the overexpression of TWG-substituted AtDGCR14L mutant did not change salt stress tolerance or pre-mRNA splicing of stress/ABA-responsive genes. Additionally, SWITCH3A (SWI3A) is a core subunit of the SWI/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes. We found that SWI3A, whose splicing depends on AtDGCR14L, actively enhances salt stress tolerance. These results revealed that AtDGCR14L may play an essential role in crosstalk between plant salt-stress response and pre-mRNA splicing mechanisms. We also unveiled the potential role of SWI3A in controlling salt stress tolerance. The TWG motif in the intrinsically disordered region of AtDGCR14L is highly conserved and crucial for DGCR14 functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Camelina circRNA landscape: Implications for gene regulation and fatty acid metabolism

Abstract Circular RNAs (circRNAs) are closed‐loop RNAs forming a covalent bond between their 3′ and 5′ ends, the back splice junction (BSJ), rendering them resistant to exonucleases and thus more stable compared to linear RNAs. Identification of circRNAs and distinction from their cognate linear RNA is only possible by sequencing the BSJ that is unique to the circRNA. CircRNAs are involved in the regulation of their cognate RNAs by increasing transcription rates, RNA stability, and alternative splicing. We have identified circRNAs from C. sativa that are associated with the regulation of germination, light response, and lipid metabolism. We sequenced light‐grown and etiolated seedlings after 5 or 7 days post‐germination and identified a total of 3447 circRNAs from 2763 genes. Most circRNAs originate from a single homeolog of the three subgenomes from allohexaploid camelina and correlate with higher ratios of alternative splicing of their cognate genes. A network analysis shows the interactions of select miRNA:circRNA:mRNAs for regulation of transcript stabilities where circRNA can act as a competing endogenous RNA. Several key lipid metabolism genes can generate circRNA, and we confirmed the presence of KASII circRNA as a true circRNA. CircRNA in camelina can be a novel target for breeding and engineering efforts.

Utley, Delecia [Department of Plant and Microbial ↗
DOE OSTI2020

Post-Transcriptional Mechanisms Respond Rapidly to Ecologically Relevant Thermal Fluctuations During Temperature-Dependent Sex Determination

An organism’s ability to integrate transient environmental cues experienced during development into molecular and physiological responses forms the basis for adaptive shifts in phenotypic trajectories. During temperature-dependent sex determination (TSD), thermal cues during discrete periods in development coordinate molecular changes that ultimately dictate sexual fate and contribute to patterns of inter- and intra-sexual variation. How these mechanisms interface with dynamic thermal environments in nature remain largely unknown. By deploying thermal loggers in wild nests of the American alligator (Alligator mississippiensis) over two consecutive breeding seasons, we observed that 80% of nests exhibit both male- and female-promoting thermal cues during the thermosensitive period, and of these nests, all exhibited both male- and female-promoting temperatures within the span of a single day. These observations raise a critical question—how are opposing environmental cues integrated into sexually dimorphic transcriptional programs across short temporal scales? To address this question, alligator embryos were exposed to fluctuating temperatures based on nest thermal profiles and sampled over the course of a daily thermal fluctuation. We examined the expression dynamics of upstream genes in the temperature-sensing pathway and find that post-transcriptional alternative splicing and transcript abundance of epigenetic modifier genes JARID2 and KDM6B respond rapidly to thermal fluctuations while transcriptional changes of downstream effector genes, SOX9 and DMRT1, occur on a delayed timescale. Our findings reveal how the basic mechanisms of TSD operate in an ecologically relevant context. We present a hypothetical hierarchical model based on our findings as well as previous studies, in which temperature-sensitive alternative splicing incrementally influences the epigenetic landscape to affect the transcriptional activity of key sex-determining genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for A Role for Differential Rubisco Activase Isoform Expression in C4 Bioenergy Grasses at High Temperature

Rubisco activase (Rca) facilitates the release of sugar-phosphate inhibitors at Rubisco catalytic sites during CO2 fixation. Most plant species express two Rca isoforms, the larger Rca-α and the shorter Rca-β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C3 plants. However, the functional role of Rca in C4 plants where Rubisco and Rca are located in a much higher [CO2] compartment is less clear. In this study, we selected four C4 bioenergy grasses and the model C4 grass setaria ( Setaria viridis ) to investigate the role of Rca in C4 photosynthesis. All five C4 grass species contained two Rca genes, one encoding Rca-α and the other Rca-β, which were positioned closely together in the genomes. A variety of abiotic stress-related motifs were identified in the Rca-α promoter of each grass, and while the Rca-β gene was constantly highly expressed at ambient temperature, Rca-α isoforms were expressed only at high temperature but never surpassed 30% of Rca-β content. The pattern of Rca-α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C4 grasses. In sorghum ( Sorghum bicolor ), sugarcane ( Saccharum officinarum ), and setaria, the induction rate of Rca-α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca-α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca-α has a functional thermo-protective role in carbon fixation in C4 grasses by sustaining Rubisco activation at high temperature.

Genomics↗
DOE OSTI2020

Decoding co-/post-transcriptional complexities of plant transcriptomes and epitranscriptome using next-generation sequencing technologies

Next-generation sequencing (NGS) technologies - Illumina RNA-seq, Pacific Biosciences isoform sequencing (PacBio Iso-seq), and Oxford Nanopore direct RNA sequencing (DRS) - have revealed the complexity of plant transcriptomes and their regulation at the co-/post-transcriptional level. Global analysis of mature mRNAs, transcripts from nuclear run-on assays, and nascent chromatin-bound mRNAs using short as well as full-length and single-molecule DRS reads have uncovered potential roles of different forms of RNA polymerase II during the transcription process, and the extent of co-transcriptional pre-mRNA splicing and polyadenylation. These tools have also allowed mapping of transcriptome-wide start sites in cap-containing RNAs, poly(A) site choice, poly(A) tail length, and RNA base modifications. The emerging theme from recent studies is that reprogramming of gene expression in response to developmental cues and stresses at the co-/post-transcriptional level likely plays a crucial role in eliciting appropriate responses for optimal growth and plant survival under adverse conditions. Although the mechanisms by which developmental cues and different stresses regulate co-/post-transcriptional splicing are largely unknown, a few recent studies indicate that the external cues target spliceosomal and splicing regulatory proteins to modulate alternative splicing. In this review, we provide an overview of recent discoveries on the dynamics and complexities of plant transcriptomes, mechanistic insights into splicing regulation, and discuss critical gaps in co-/post-transcriptional research that need to be addressed using diverse genomic and biochemical approaches.

Biochemistry & Molecular Biology↗
DOE OSTI2020

An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells

Proinflammatory cytokines are important mediators of pancreatic beta cell dysfunction and demise in the early stages of type 1 diabetes (T1D). Interferon-a (IFNa), a type I interferon member, is expressed in the islets of T1D individuals and it is expression and signaling is regulated by both genetic (T1D risk variants) and environmental factors (viral infections) associated to T1D. We presently characterized human beta cells responses to IFNa by combining ATAC-seq, RNA-seq and proteomics assays. The initial beta cell response to IFNa was characterized by major chromatin remodeling, followed by marked changes in transcriptional and translational regulation. IFNa-induced changes in alternative splicing (AS) and first exon usage increased the diversity of transcripts expressed by beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may significantly expand the peptide repertoire presented by beta cells to the immune system. On the other hand, beta cells up-regulated checkpoint proteins, such as PDL1 and HLA-E, that may protect them against the autoimmune assault. Data mining of the present multi-omics analysis led to the identification of two compound classes that revert IFNa effects on human beta cells and may be translated to clinical trials.

60 APPLIED LIFE SCIENCES↗