Biology subjects

Hrabeta-Robinson, E.

Publications and source records attributed to Hrabeta-Robinson, E..

6 recordsLinked to original sources

bioRxiv2024

U2AF1 S34F enhances tumorigenic potential of lung cells by exhibiting synergy with KRAS mutation and altering response to environmental stress

Although U2AF1S34F is a recurrent splicing factor mutation in lung adenocarcinoma (ADC), U2AF1S34F alone is insufficient for producing tumors in previous models. Because lung ADCs with U2AF1S34F frequently have co-occurring KRAS mutations and smoking histories, we hypothesized that tumor-forming potential arises from U2AF1S34F interacting with oncogenic KRAS and environmental stress. To elucidate the effect of U2AF1S34F co-occurring with a second mutation, we generated human bronchial epithelial cells (HBEC3kt) with co-occurring U2AF1S34F and KRASG12V. Transcriptome analysis revealed that co-occurring U2AF1S34F and KRASG12V differentially impacts inflammatory, cell cycle, and KRAS pathways. Subsequent phenotyping found associated suppressed cytokine production, increased proliferation, anchorage-independent growth, and tumors in mouse xenografts. Additionally, HBEC3kts harboring U2AF1S34F alter splicing in stress granule protein genes, show increased stress granule formation, and have increased viability in cigarette smoke concentrate. Our results suggest that U2AF1S34F may potentiate transformation by granting precancerous cells survival advantage in environmental stress, permitting accumulation of additional mutations, like in KRAS, which synergize with U2AF1S34F to transform the cell.

cancer biology↗

bioRxiv2024

Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing

Studies of in vivo chromatin organization have relied on the accessibility of the underlying DNA to nucleases or methyltransferases, which is limited by their requirement for purified nuclei and enzymatic treatment. Here, we introduce a nanopore-based sequencing technique called Small-Molecule Adduct sequencing (SMAdd-seq), where we profile chromatin accessibility by treating nuclei or intact cells with a small molecule, angelicin. Angelicin reacts with thymine bases in linker DNA not bound to core nucleosomes after UV light exposure, thereby labeling accessible DNA regions. By applying SMAdd-seq in Saccharomyces cerevisiae, we demonstrate that angelicin-modified DNA can be detected by its distinct nanopore current signals. To systematically identify angelicin modifications and analyze chromatin structure, we developed a neural network model, NEural network for mapping MOdifications in nanopore long-reads (NEMO). NEMO accurately called expected nucleosome occupancy patterns near transcription start sites at both bulk and single-molecule levels. We observe heterogeneity in chromatin structure and identify clusters of single-molecule reads with varying configurations at specific yeast loci. Furthermore, SMAdd-seq performs equivalently on purified yeast nuclei and intact cells, indicating the promise of this method for in vivo chromatin labeling on long single molecules to measure native chromatin dynamics and heterogeneity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/585815v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@14a0f8dorg.highwire.dtl.DTLVardef@143030eorg.highwire.dtl.DTLVardef@7d8ed4org.highwire.dtl.DTLVardef@12c3b05_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Detecting haplotype-specific transcript variation in long reads with FLAIR2

BackgroundRNA-Seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme which mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung ADC cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, short read RNA-Seq has limited the communitys ability to interrogate both forms of RNA variation simultaneously. ResultsWe employed long-read technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We have developed a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generated nanopore data with high sequence accuracy of H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We applied our workflow to identify key inosine-isoform associations to help clarify the prominence of ADAR in tumorigenesis. ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns. HighlightsO_LIFLAIR2 has improved transcript isoform detection and incorporates sequence variants for haplotype-specific transcript detection. C_LIO_LIIn addition to haplotype-specific variant detection, it identifies transcript-specific RNA editing C_LIO_LIAble to identify haplotype-specific transcript isoform bias in expression C_LIO_LILong-read sequencing identifies hyperedited transcripts that are missed from short-read sequencing methods for a more comprehensive identification of ADAR targets C_LI

bioinformatics↗

bioRxiv2019

Nanopore sequencing reveals U2AF1 S34F-associated full-length isoforms

U2AF1 S34F is one of the most recurrent splicing factor mutations in lung adenocarcinoma (ADC) and has been shown to cause transcriptome-wide pre-mRNA splicing alterations. While U2AF1 S34F-associated splicing alterations have been described, the function of altered mRNA isoform changes remains largely unexplored. To better understand the impact U2AF1 S34F has on isoform fate and function, we conducted high-throughput long-read cDNA sequencing from isogenic human bronchial epithelial cells with and without U2AF1 S34F mutation. We found that nearly 75% (49,366) of our long-read constructed multiexon isoforms do not overlap GENCODE or short-read assembled isoforms. We found 198 transcript isoforms with significant expression and usage changes caused by U2AF1 S34F mutation, including a novel lncRNA. Isoforms from immune-related genes were largely downregulated in mutant cells, none of which were found to have splicing changes. Finally, isoforms likely targeted by nonsense-mediated decay were preferentially downregulated in U2AF1 S34F cells, suggesting that the impact of observed isoform changes may alter the translational output of affected genes. Altogether, long-read sequencing provided additional insights into transcriptome alterations and downstream functional consequences associated with U2AF1 S34F mutation.

genomics↗

bioRxiv2018

A two-step probing method to compare lysine accessibility across macromolecular complex conformations

Structural models of multi-megadalton molecular complexes are appearing in increasing numbers, in large part because of technical advances in cryo-electron microscopy realized over the last decade. However, the inherent complexity of large biological assemblies comprising dozens of components often limits the resolution of structural models. Furthermore, multiple functional configurations of a complex can leave a puzzle as to how one intermediate moves to the next stage. Orthogonal biochemical information is crucial to understanding the molecular interactions that drive those rearrangements. We present a two-step method for chemical probing detected by tandem mass-spectrometry to globally assess the reactivity of lysine residues within purified macromolecular complexes. Because lysine side chains often balance the negative charge of RNA in ribonucleoprotein complexes, the method is especially powerful for detecting changes in protein-RNA interactions. Probing the E. coli 30S ribosome subunit showed that the reactivity pattern of lysine residues quantitatively reflects structure models from X-ray crystallography. We assessed differences in two conformations of purified human spliceosomes. Our results demonstrate that this method supplies powerful biochemical information that aids in functional interpretation of atomic models of macromolecular complexes at the intermediate resolution often provided by cryo-electron microscopy.

molecular biology↗

bioRxiv2018

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 have been identified with short-read RNA sequencing, a critical barrier in understanding the functional consequences of these splicing changes is that we lack the full transcript context in which these changes are occurring. Using nanopore sequencing technology, we have resequenced full-length cDNA from CLL samples with and without the hotspot SF3B1 K700E mutation, and a normal B cell. We have developed a workflow called FLAIR (Full-Length Alternative Isoform analysis of RNA), leveraging the full-length transcript sequencing data that nanopore affords. We report results from nanopore sequencing that are concordant with known SF3B1 biology from short read sequencing as well as altered intron retention events more confidently observed using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E samples identifies alternative upstream 3 splice sites associated with SF3B1K700E. We also find downregulation of intron retention events in SF3B1K700E relative to SF3B1WT and no difference between CLL SF3B1MT and B cell, suggesting an aberrant intron retention landscape in CLL samples lacking SF3B1 mutation. With full-length isoforms, we are able to better estimate the abundance of RNA transcripts that are productive and will likely be translated versus those that are unproductive. Validation from short-read data also reveals a strong branch point sequence in these downregulated intron retention events, consistent with previously reported branch points associated with mutated SF3B1. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

genomics↗