Cooperation of TRAMP cofactors was study combining CRAC and RNAseq approach. CRAC Samples were tagged (HTP for all except Sample4 which carry an Flag-Prescission-HIs6). Duplicate CRAC experiments were carried out on each protein. RNAseq was carried out on wt and ∆trf5 stains (4 and 3 repeats respectively) using Lexogen SENSE mRNA kit standard protocol.
During nuclear surveillance, the RNA exosome functions together with the TRAMP complexes, which include the RNA helicase Mtr4 together with a RNA binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions by TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. Comparison of the binding sites of TRAMP components with multiple nuclear RNA binding proteins revealed preferential colocalization of subsets of factors. On pre-mRNAs, ∆trf5 deletion caused reduced Mtr4 binding and RNA accumulation of Trf5 top targets.
Substrate Specificity of the TRAMP Nuclear Surveillance Complexes.,Delan-Forino C, Spanos C, Rapsilber J, Tollervey D,. - Tollervey Lab, University of Edinburgh - WTCCB. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE135526