Biology subjects

Shefer, K.

Publications and source records attributed to Shefer, K..

2 recordsLinked to original sources

bioRxiv2026

A Synthetic Mirtron Platform Enables Stable and Robust Splicing-Dependent Gene Silencing in Plants

Post-transcriptional gene silencing (PTGS) is widely used for gene function studies and crop improvement; however, conventional transgene-based RNAi and artificial microRNA (amiRNA) approaches are often subject to transgene self-silencing, epigenetic inactivation, viral suppressors of RNA silencing, and regulatory complexity. Here, we establish for the first time a synthetic mirtron platform in plants that defines a splice-gated, non-canonical PTGS architecture, mechanistically distinct from existing RNAi strategies. We demonstrate that precise intron splicing and lariat debranching are essential for target gene silencing, directly coupling pre-mRNA splicing to small RNA-mediated regulation. An optimized mirtron mediates efficient, stable, and heritable silencing of PHYTOENE DESATURASE in Arabidopsis thaliana and enables multiplex silencing of endogenous AUXIN RESPONSE FACTORS in potato, demonstrating applicability in a major crop species. Moreover, mirtron-mediated gene silencing remains highly effective in the presence of the viral suppressor P19, unlike canonical amiRNA-based silencing, highlighting its greater resistance to viral suppression and utility for host-induced gene silencing. Because mirtrons are embedded within endogenous introns, they can be co-expressed with host genes, inheriting their native spatial and temporal expression patterns. When precisely introduced, this architecture supports regulatory outcomes similar to those of gene-edited products that lack foreign DNA. Together, these findings define mirtrons as a compact, stable, and application-ready PTGS platform useful for studying gene regulation and crop biotechnology.

plant biology↗

bioRxiv2020

A Novel Role for Nucleolin in Splice Site Selection

Latent 5 splice sites are highly abundant in human introns, yet, are apparently not normally used. Splicing at most of these sites would incorporate in-frame stop codons generating nonsense mRNAs. Importantly, under stress and in cancer, splicing at latent sites is activated generating nonsense mRNAs from thousands of genes. Previous studies point to an unresolved RNA quality control mechanism that suppresses latent splicing independently of NMD. They further demonstrated a pivotal role for initiator-tRNA in this mechanism, through its interaction with the AUG codon, independent of its role in protein translation. To further elucidate this mechanism, here we searched for nuclear proteins directly bound to initiator-tRNA in the nucleus. We identified nucleolin (NCL), a multifunctional, abundant, and conserved protein, as a novel regulator of splice site selection. Starting with UV crosslinking, we show that NCL is directly and specifically interacting with initiator-tRNA in the nucleus, but not in the cytoplasm. In support of NCL involvement in this mechanism, we show activation of latent splicing in hundreds of transcripts upon NCL knockdown, disrupting gene transcripts involved in several important cellular pathways and cell metabolism functions (e.g. transcription factors, oncogenes, kinases, splicing factors, translation factors, and genes affecting cell motility, proliferation, and cellular trafficking). We thus propose NCL, a component of the endogenous spliceosome, through its direct interaction with initiator-tRNA and its effect on latent splicing as the first documented protein of a nuclear quality control mechanism that regulates splice site selection to protect cells from latent splicing that would generate defective mRNAs.

genomics↗