Biology subjects

Lackey, L.

Publications and source records attributed to Lackey, L..

6 recordsLinked to original sources

bioRxiv2026

Divergent RNA structures support accurate splicing of the SF3B1-sensitive MAP3K7 intron

Splicing is governed by interactions between the spliceosome and precursor RNA sequence and structural elements. However, the relative contributions of RNA sequence and structural elements remain unclear. Here, we systematically dissect these determinants using a high-throughput mutagenesis approach with the MAP3K7 intron reporter. The MAP3K7 gene encodes a serine/threonine kinase involved in response to environmental stress. MAP3K7 precursor RNA contains a cryptic 3 splice site that increases in use when the core spliceosomal protein SF3B1 is mutated. SF3B1 mutations are known to promote aberrant splicing and are associated with cancer, particularly the lysine 700 to glutamate mutation (K700E). We designed a pooled library of 249 MAP3K7 mutants targeting branch points, RNA-binding protein motifs, nucleotide composition and predicted structural elements. The impact of these mutants on splicing was measured in the context of normal and SF3B1 K700E expression. RNA structure was assessed in parallel using in vitro high-throughput SHAPE-MAP chemical probing. We found that branchpoint mutations drive the strongest increases in cryptic splice-site use. There is no overall correlation between cryptic splice-site use and structural similarity to the wild-type MAP3K7 RNA. However, mutants within an RNA binding protein hotspot (containing U2AF2, U2AF1, KHSRP and SRSF2 sites) are associated with cryptic splice-site use and structural similarity to wild-type MAP3K7 RNA. These structural changes are associated with increased ensemble diversity. Our results demonstrate that although there are key structured regions within an RNA, there is also extensive variability where divergent RNA structures allow for accurate splicing.

molecular biology↗

bioRxiv2026

Neural microexons contain lengthened sequence and extended RNA structure between the branchpoint and splice site motif

Microexons are short exons that are highly conserved in vertebrates and are essential for neurodevelopment. Their small size poses a challenge for regulatory protein binding and exon-definition splice site recognition, which typically relies on standard length exons. Here, we determine the sequence and RNA structural features of neural microexons in humans and in the chick developmental model organism. We demonstrate that a subset of neural microexons undergoes dynamic, stage-specific regulation during chick embryonic brain development that correlates with expression of known microexon regulators, SRRM4 and NOVA1. Using experimental RNA structure-probing on a subset of neural microexons, we show that shared RNA secondary structures between orthologous human and chicken microexon precursor mRNAs primarily occur in regions of high sequence conservation. We find that both human and chicken neural microexons have extended functional distance between the branchpoint and the 3 splice. Structurally, branchpoint-to-splice site regions are unusually accessible and relatively unpaired compared to other exon classes. Our data suggest that microexon splicing relies on structural accessibility of the branch-point-to-splice site region, which may influence accessibility for SRRM4 binding and alleviate steric constraints for spliceosome assembly. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/699960v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@18d1f65org.highwire.dtl.DTLVardef@854fforg.highwire.dtl.DTLVardef@10463a1org.highwire.dtl.DTLVardef@128e7c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Precursor RNA structural patterns at SF3B1 mutation sensitive cryptic 3' splice sites

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3 splice site selection. SF3B1 mutation is common in myelodysplastic syndrome and other blood disorders. The most common mutation in SF3B1 is K700E, a lysine to glutamic acid change within the pre-mRNA interacting heat repeat domain. A hallmark of SF3B1 mutation is an increased use of cryptic 3 splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 core splice junctions that are mis-spliced with SF3B1 K700E mutation. We use our core set to test whether SF3B1-sensitive splice sites are different from control cryptic 3 splice sites via RNA structural accessibility. As a comparison, we define a set of SF3B1-resistant splice junctions with cryptic splice site use that does not change with SF3B1 K700E mutation. We find sequence differences between SF3B1-sensitive and SF3B1-resistant junctions, particularly at the cryptic sites. SF3B1-sensitive cryptic 3 splice sites are within an extended polypyrimidine tract and have lower splice site strength scores. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3 splice sites is similar. In addition, this pattern can be found in both SF3B1-resistant and SF3B1-sensitive junctions. However, SF3B1-sensitive junctions have cryptic splice sites that are less structurally distinct from the canonical splice sites. In addition, SF3B1-sensitive splice junctions are overall more flexible than SF3B1-resistant junctions. Our results suggest that the SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3 splice site recognition in the presence of SF3B1 mutation.

molecular biology↗

bioRxiv2024

Altered polyadenylation site usage in SERPINA1 3'UTR in response to cellular stress affects A1AT protein expression

Alternative polyadenylation results in different 3 isoforms of messenger RNA (mRNA) transcripts. Alternative polyadenylation in the 3 untranslated region (3UTR) can alter RNA localization, stability and translational efficiency. The SERPINA1 mRNA has two distinct 3 UTR isoforms, both of which express the protease inhibitor -1-antitrypsin (A1AT). A1AT is an acute phase protein that is expressed and secreted from liver hepatocytes and upregulated during inflammation. Low levels of A1AT in the lung contributes to chronic obstructive pulmonary disease, while misfolding of A1AT in the liver contributes to liver cirrhosis. We analyzed the dynamics of alternative polyadenylation during cellular stress by treating the liver cell line HepG2 with the cytokine interleukin 6 (IL-6), ethanol or peroxide. SERPINA1 is transcriptionally upregulated after IL-6 treatment and has altered polyadenylation, resulting in an increase in long 3UTR isoforms. We find that the long 3UTR represses endogenous A1AT protein expression even with high levels of SERPINA1 mRNA. SERPINA1 expression and 3 end processing were not affected by ethanol or peroxide. IL-6-induced changes in transcriptome-wide transcriptional regulation suggest changes to the endoplasmic reticulum and in secretory protein processing. Our data suggest that inflammation influences polyA site choice for SERPINA1 transcripts, resulting in reduced A1AT protein expression.

molecular biology↗

bioRxiv2021

Quantitative prediction of variant effects on alternative splicing using endogenous pre-messenger RNA structure probing

Splicing is a highly regulated process that depends on numerous factors. It is particularly challenging to quantitatively predict how a mutation will affect precursor messenger RNA (mRNA) structure and the subsequent functional consequences. Here we use a novel Mutational Profiling (-MaP) methodology to obtain highly reproducible endogenous precursor and mature mRNA structural probing data in vivo. We use these data to estimate Boltzmann suboptimal ensembles, and predict the structural consequences of mutations on precursor mRNA structure. Together with a structural analysis of recent cryo-EM spliceosome structures at different stages of the splicing cycle, we determined that the footprint of the Bact complex on precursor mRNA is best able to predict splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene. However, structure alone only achieves 74% accuracy. We therefore developed a {beta}-regression weighting framework that incorporates splice site strength, structure and exonic/intronic splicing regulatory elements which together achieves 90% accuracy for 47 known and six newly discovered splice-altering variants. This combined experimental/computational framework represents a path forward for accurate prediction of splicing related disease-causing variants.

genomics↗

bioRxiv2019

A rabbit monoclonal antibody against the antiviral and cancer genomic DNA mutating enzyme APOBEC3B

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B through a series of immunoblot, immunofluorescence microscopy, and immunohistochemistry experiments. Positive results indicate that one mAb, 5210-87-13, will be useful for fundamental research in virology and cancer biology and that immunohistochemical quantification of A3B enzyme levels may constitute a clinical biomarker for tumor evolvability and differential treatment.

cancer biology↗