Tobramycin affinity tag purification of spliceosomes.

ABSTRACT

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