In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes
The RNA exosome is a multisubunit protein complex that exhibits a 3′ to 5′ exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.
Key wordsRNA exosome Cap labeling of mRNA In vitro reconstitution of mammalian ribosomal complexes Toeprinting GTPBP1 mRNA degradation assay
This work was supported by NIH grant GM80623 to T.V.P. and NIH grant AI123406 to C.U.T.H.
- 21.Schmidt C, Kowalinski E, Shanmuganathan V, Defenouillère Q, Braunger K, Heuer A, Pech M, Namane A, Berninghausen O, Fromont-Racine M, Jacquier A, Conti E, Becker T, Beckmann R (2016) The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Science 354:1431–1433PubMedCrossRefPubMedCentralGoogle Scholar
- 32.Lomakin IB, Hellen CU, Pestova TV (2000) Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol Cell Biol 20:6019–6029PubMedPubMedCentralCrossRefGoogle Scholar
- 38.Unbehaun A, Borukhov SI, Hellen CU, Pestova TV (2004) Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP. Genes Dev 18:3078–3093PubMedPubMedCentralCrossRefGoogle Scholar
- 44.Anthony DD, Merrick WC (1992) Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J Biol Chem 1267:1554–1562Google Scholar