Biology subjects

Shilo, A.

Publications and source records attributed to Shilo, A..

3 recordsLinked to original sources

bioRxiv2022

HiFENS: High-throughput FISH detection of endogenous pre-mRNA splicing isoforms

Splicing factors play an essential role in regulation of alternative pre-mRNA splicing. While much progress has been made in delineating the mechanisms of the splicing machinery, the identity of signal transduction pathways and upstream factors that regulate splicing factor activity is less well understood. A major challenge in the discovery of upstream regulatory factors of pre-mRNA splicing is the scarcity of functional genomics screening methods to monitor splicing outcomes of endogenous genes. Here, we have developed HiFENS (High throughput FISH Detection of ENdogenous Splicing Isoforms), a high-throughput imaging assay based on hybridization chain reaction (HCR) and used HiFENS to screen for cellular factors that regulate alternative splicing of endogenous genes. We demonstrate optimized detection with high specificity of endogenous splicing isoforms and multiplexing of probes for accurate detection of splicing outcomes with single cell resolution. As proof-of-principle, we perform an RNAi screen of 702 human kinases and identify potential candidate upstream splicing regulators of the FGFR2 gene. HiFENS should be a useful tool for the unbiased delineation of cellular pathways involved in alternative splicing regulation.

cell biology↗

bioRxiv2022

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

cancer biology↗

bioRxiv2018

Interplay of primary sequence and RNA secondary structure in determining 5′ splice site choice

Selective use of 5' splice sites is a common mechanism by which pre-mRNAs are alternatively spliced. Whereas the sequence requirements of 5' splice site choice have been well characterized, other important determinants remain poorly defined. Here we apply a combination of structural mapping by SHAPE-MaP and targeted mutational analysis in a cell-based system to comprehensively probe the interplay of primary sequence, secondary RNA structure, regulatory elements and linear splice site position to determine mechanisms of splice site choice in vivo. Using the disease-causing alternative 5' splice site selection in LMNA in the premature aging disorder Hutchinson-Gilford Progeria Syndrome as a model system, we identify RNA secondary structural elements near the alternative 5' splice sites. We show that splice site choice is significantly influenced by the structural context of the available splice sites. While local structure alone is not sufficient to account for splice site selection, the choice of 5' splice sites depends on the structural stability of the 5' splice site region which is conferred by downstream elements. In addition, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5' splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results reveal an intricate interplay amongst RNA sequence, secondary structure and splice site position in determining 5' splice site choice.

molecular biology↗