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DOE OSTI2024

RNA Splicing Events in Circulation Distinguish Individuals With and Without New-onset Type 1 Diabetes

Context: Alterations in RNA splicing may influence protein isoform diversity that contributes to or reflects the pathophysiology of certain diseases. Whereas specific RNA splicing events in pancreatic islets have been investigated in models of inflammation in vitro, how RNA splicing in the circulation correlates with or is reflective of type 1 diabetes (T1D) disease pathophysiology in humans remains unexplored. Objective: To use machine learning to investigate if alternative RNA splicing events differ between individuals with and without new-onset T1D and to determine if these splicing events provide insight into T1D pathophysiology. Methods: RNA deep sequencing was performed on whole blood samples from 2 independent cohorts: a training cohort consisting of 12 individuals with new-onset T1D and 12 age- and sex-matched nondiabetic controls and a validation cohort of the same size and demographics. Machine learning analysis was used to identify specific isoforms that could distinguish individuals with T1D from controls. Results: Distinct patterns of RNA splicing differentiated participants with T1D from unaffected controls. Notably, certain splicing events, particularly involving retained introns, showed significant association with T1D. Machine learning analysis using these splicing events as features from the training cohort demonstrated high accuracy in distinguishing between T1D subjects and controls in the validation cohort. Gene Ontology pathway enrichment analysis of the retained intron category showed evidence for a systemic viral response in T1D subjects. Conclusion: Alternative RNA splicing events in whole blood are significantly enriched in individuals with new-onset T1D and can effectively distinguish these individuals from unaffected controls. Further, our findings also suggest that RNA splicing profiles offer the potential to provide insights into disease pathogenesis.

60 APPLIED LIFE 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

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 OSTI2025

Mutation-driven RRE stem-loop II conformational change induces HIV-1 nuclear export dysfunction

Abstract The Rev response element (RRE) forms an oligomeric complex with the viral protein Rev to facilitate the nuclear export of intron-retaining viral RNAs during the late phase of HIV-1 (human immunodeficiency virus type 1) infection. However, the structures and mechanisms underlying this process remain largely unknown. Here, we determined the crystal structure of the HIV-1 RRE stem-loop II (SLII), revealing a unique three-way junction architecture in which the base stem (IIa) bifurcates into the stem-loops (IIb and IIc) to compose Rev binding sites. The crystal structures of various SLII mutants demonstrated that while some mutants retain the same “compact” fold as the wild type, other single-nucleotide mutants induce drastic conformational changes, forming an “extended” SLII structure. Through in vitro Rev binding assays and Rev activity measurements in HIV-1-infected cells using structure-guided SLII mutants designed to favor specific conformers, we showed that while the compact fold represents a functional SLII, the alternative extended conformation inhibits Rev binding and oligomerization and consequently stimulates HIV-1 RNA nuclear export dysfunction. The propensity of SLII to adopt multiple conformations as captured in crystal structures and their influence on Rev oligomerization illuminate emerging perspectives on RRE structural plasticity-based regulation of HIV-1 nuclear export and provide opportunities for developing anti-HIV drugs targeting specific RRE conformations.

Biochemistry & Molecular Biology↗

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

Webb-Robertson, Bobbie-Jo M↗

DOE OSTI2022

Silencing of Dicer‐like protein 2a restores the resistance phenotype in the rice mutant, sxi4 ( suppressor of Xa21‐mediated immunity 4 )

SUMMARY The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae ( Xoo ), and upon recognition of the RaxX21‐sY peptide produced by Xoo , XA21 activates the plant immune response. Here we screened 21 000 mutant plants expressing XA21 to identify components involved in this response, and reported here the identification of a rice mutant, sxi4, which is susceptible to Xoo. The sxi4 mutant carries a 32‐kb translocation from chromosome 3 onto chromosome 7 and displays an elevated level of DCL2a transcript, encoding a Dicer‐like protein . Silencing of DCL2a in the sxi4 genetic background restores resistance to Xoo . RaxX21‐sY peptide‐treated leaves of sxi4 retain the hallmarks of XA21‐mediated immune response. However, WRKY45‐1 , a known negative regulator of rice resistance to Xoo , is induced in the sxi4 mutant in response to RaxX21‐sY peptide treatment. A CRISPR knockout of a short interfering RNA (TE‐siRNA815) in the intron of WRKY45‐1 restores the resistance phenotype in sxi4 . These results suggest a model where DCL2a accumulation negatively regulates XA21‐mediated immunity by altering the processing of TE‐siRNA815.

Liu, Furong↗