GSE105471
A method for the acute and rapid degradation of endogenous protein
We developed Trim-Away as a a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away is able to remove unmodified native proteins within minutes of application by making use of the TRIM21 ubiquitin ligase that regognises antibody-protein complexes and harnesses the cellular degradation machinery. We performed transcriptome profiling (RNAseq) on NIH3T3 and NIH3T3-mCherry-mTRIM21 cells  following electroporation with non-targeting antibody (IgG), bovine serum albumin (BSA), or buffer (PBS). We found that a total of only 8 protein-encoding transcripts were consistently downregulated more than 2-fold in the NIH3T3-mCherry-mTRIM21 cells (Kdm5d, Ddx3y, Eif2s3y, Uty, Asb4, Fat4, Papss2 and Lama2), although we cannot rule out the possibility that this is an indirect consequence of lentivirus construct integration rather than a direct consequence of TRIM21 overexpression. Notably, transcripts encoding previously described ligands of TRIM21, IRF-3, IRF-5, Skp2, DAXX, DDX41, and SQSTM1 were not significantly increased or decreased upon overexpression of TRIM21. Our experiments suggest that the cells overexpressing TRIM21 behave similar to wild-types. Overall design: mRNA profiles of NIH3T3 and NIH3T3-mCherry-mTRIM21 cells following electroporation with antibody (IgG), BSA or PBS were generated by deep sequencing, in triplicate, using a Illumina NextSeq 500 platform.