Pre-mRNA transcripts become rapidly associated with many RNA-binding proteins, including hnRNP proteins, cap-binding proteins, SR proteins, etc; in the test tube this binding does not require splice sites or ATP. The E complex, or early complex, is the first detectable functional intermediate in spliceosome assembly in vitro. It is an ATP-independent complex. When a functional 5' splice site is present, it is bound by the U1 snRNP. The splicing factor U2AF (65 and 35 kDa subunits) binds to the polypyrimidine tract (Y)n and the AG dinucleotide at the 3' splice site, respectively. SF1/mBBP binds to the branch site. Binding of many of these factors is cooperative; e.g., SR proteins and U2AF apparently interact with each other, facilitating their binding to the pre-mRNA. In the presence of ATP, the E complex is converted to the first ATP-dependent spliceosomal complex, namely the A complex.