Publications by Year: Forthcoming

Background Changes in RNA splicing over the course of evolution have profoundly diversified the functional landscape of the human genome. While DNA sequences proximal to intron-exon junctions are known to be critical for RNA splicing, the impact of distal intronic sequences remains underexplored. Emerging evidence suggests that inverted pairs of intronic Alu elements can promote exon skipping by forming RNA stem-loop structures. However, their prevalence and influence throughout evolution remain unknown.

Results Here, we present a systematic analysis of inverted Alu pairs across the human genome to assess their impact on exon skipping through predicted RNA stem-loop formation and their relevance to hominoid evolution. We found that inverted Alu pairs, particularly pairs of AluY-AluSx1 and AluSz-AluSx, are enriched in the flanking regions of skippable exons genome-wide and are predicted to form stable stem-loop structures. Exons defined by weak 3′ acceptor and strong 5′ donor splice sites appear especially prone to this skipping mechanism. Through comparative genome analysis across nine primate species, we identified 67,126 hominoid-specific Alu insertions, primarily from AluY and AluS subfamilies, which form inverted pairs enriched across skippable exons in genes of ubiquitination-related pathways. Experimental validation of exon skipping among several hominoid-specific inverted Alu pairs further reinforced their potential evolutionary significance.

Conclusion This work extends our current knowledge of the roles of RNA secondary structure formed by inverted Alu pairs and details a newly emerging mechanism through which transposable elements have contributed to genomic innovation across hominoid evolution at the transcriptomic level.

Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) are common neurodegenerative disorders for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 429 neurons from three C9ORF72 ALS, six C9ORF72 FTD, seven AD, and twenty-three neurotypical control brains revealed significantly increased burdens in somatic single nucleotide variant (sSNV) and insertion/deletion (sIndel) in all three disease conditions. Mutational signature analysis identified a disease-associated sSNV signature suggestive of oxidative damage and an sIndel process, affecting 28% of ALS, 79% of FTD, and 65% of AD neurons but only 5% of control neurons (diseased vs. control: OR=31.20, p=2.35X10^-10). Disease-associated sIndels were primarily two-basepair deletions resembling signature ID4, which was previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Duplex sequencing confirmed the presence of sIndels and identified similar single-strand events as potential precursor lesions. TOP1-associated sIndel mutagenesis and resulting genome instability may thus represent a common mechanism of neurodegeneration.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-β and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher ( two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.