ABSTRACT
Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as alternative splicing and mRNA translation, implicated in several developmental processes. It was previously shown that SAM68 regulates the alternative splicing of the mechanistic target of rapamycin (mTor), but the mechanism regulating this process remains elusive. Here, we report that SAM68 interacts with U1 small nuclear ribonucleoprotein (U1 snRNP) to promote splicing at the 5' splice site in intron 5 of mTor. We also show that this direct interaction is mediated through U1A, a core-component of U1snRNP. SAM68 was found to bind the RRM1 domain of U1A through its C-terminal tyrosine rich region (YY domain). Deletion of the U1A-SAM68 interaction domain or mutation in SAM68-binding sites in intron 5 of mTor abrogates U1A recruitment and 5' splice site recognition by the U1 snRNP, leading to premature intron 5 termination and polyadenylation. Taken together, our results provide the first mechanistic study by which SAM68 modulates alternative splicing decision, by affecting U1 snRNP recruitment at 5' splice sites.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , RNA Precursors/genetics , RNA Splicing , RNA-Binding Proteins/genetics , RNA/genetics , Ribonucleoprotein, U1 Small Nuclear/genetics , TOR Serine-Threonine Kinases/genetics , Adaptor Proteins, Signal Transducing/deficiency , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cell Line , Exons , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Deletion , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Introns , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , RNA/metabolism , RNA Precursors/metabolism , Ribonucleoprotein, U1 Small Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Epithelial-to-mesenchymal transition (EMT) is a process by which cancer cells gain the ability to leave the primary tumor site and invade surrounding tissues. These metastatic cancer cells can further increase their plasticity by adopting an amoeboid-like morphology, by undergoing mesenchymal-to-amoeboid transition (MAT). We found that adhering cells produce spreading initiation centers (SICs), transient structures that are localized above nascent adhesion complexes, and share common biological and morphological characteristics associated with amoeboid cells. Meanwhile, spreading cells seem to return to a mesenchymal-like morphology. Thus, our results indicate that SIC-induced adhesion recapitulates events that are associated with amoeboid-to-mesenchymal transition (AMT). We found that polyadenylated RNAs are enriched within SICs, blocking their translation decreased adhesion potential of metastatic cells that progressed through EMT. These results point to a so-far-unknown checkpoint that regulates cell adhesion and allows metastatic cells to alter adhesion strength to modulate their dissemination.
