Prespliceosome and Spliceosome Isolation and Analysis

Spliceosomes are multicomponent enzymes that remove introns from premessenger RNAs (pre-mRNAs) (1 –3 ) in the reaction known as pre-mRNA splicing. Spliceosomes are ribonucleoprotein (RNP) machines consisting of both RNA and protein components. SnRNPs, composed of small nuclear RNAs (snRNAs) and associated proteins, are critical for the pre-mRNA splicing reaction. In addition to the snRNPs there are several proteins required for splicing, some of which are components of the spliceosome (4 ). Two different spliceosomes are required to remove two classes of introns. The major class of introns usually have GU and AG dinucleotides at the 5′ and 3′ splice sites respectively, whereas the minor class of introns, the AT-AC introns, usually have AT and AC dinucleotides at the 5′ and 3′ splice sites (5 ,6 ). Here we focus our attention on the major or conventional spliceosome, which shares the U5 small ribonucleoprotein particle (snRNP) with the AT-AC spliceosome, but otherwise has a unique set of snRNPs: U1, U2, and U4/U6 (7 ).