ABSTRACT
The loss of protein homeostasis results in cytotoxic protein aggregates, a common hallmark of aging and neurological diseases. Here, we present an adjusted filter-trapping assay protocol to detect global aggregated proteins in human cell lines, via a high-sensitive protein staining method. This protocol also details an alternative approach to monitor specific protein aggregates trapped in the filter membrane, by subsequent immunoblotting of ectopically expressed and endogenous proteins. For complete details on the use and execution of this protocol, please refer to Chhipi-Shrestha et al. (2022).
Subject(s)
Protein Aggregates , Proteins , Biological Assay , Cell Line , Humans , Staining and LabelingABSTRACT
Chemical splicing modulators that bind to the spliceosome have provided an attractive avenue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. However, the biological effect of proteins containing translated intron sequences remains unclear. Here, we identify a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) via genome-wide ribosome profiling and bio-orthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through c-Jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.
Subject(s)
JNK Mitogen-Activated Protein Kinases/genetics , Mechanistic Target of Rapamycin Complex 1/genetics , RNA Splicing/genetics , Spliceosomes/genetics , Cell Line , Enzyme Inhibitors/pharmacology , Humans , Introns , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Pyrans/pharmacology , RNA Splicing/drug effects , RNA-Seq , Spiro Compounds/pharmacology , Spliceosomes/drug effectsABSTRACT
RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.
