Tobramycin Affinity Tag Purification of Spliceosomes

The ability to isolate native ribonucleoprotein (RNP) particles is of fundamental importance in the study of processes suchas pre-messenger RNA (mRNA) processing and translation. We have developed an RNA affinity tag that allows the large-scalepreparation of native spliceosomes in a solid-phase assembly scheme. A tobramycin-binding aptamer cotranscriptionally addedto the 3′ end of the pre-mRNA is used to bind the pre-mRNA to tobramycin immobilized on a matrix. Incubation of the pre-mRNAthus immobilized allows the assembly of spliceosomes, which can be released from the matrix under native conditions by competitionwith tobramycin. Further density-gradient centrifugation affords highly purified spliceosomes suitable for the characterizationof associated proteins by mass spectrometry as well as for studies using biochemical and biophysical methods. Although themethod was developed for the preparation of spliceosomes, it is likewise applicable to the preparation of other RNP particles.