Figure 1. Schematic representation of the two‐step splicing pathway of nuclear pre‐mRNA introns and the conserved sequence elements of yeast and metazoan pre‐mRNAs.
  1. Two successive phosphoester transfer reactions lead to excision of the (lariat) intron and joining of the exons (for details, see the text). The branch‐site adenosine is shown in bold; the polypyrimidine‐tract is represented by (Yn);
  2. Here, two exons are separated by an intron.
The consensus sequences in metazoans and yeast at the 5′ splice site (5′SS), branch‐site sequence (BS), and 3′ splice site (3′SS) are as indicated, where N is any nucleotide, R is a purine, and Y is a pyrimidine. The polypyrimidine‐tract (Yn) is a pyrimidine‐rich stretch located between the BS and 3′SS.

Figure 2. Protein composition of human snRNPs. The spliceosomal snRNPs are represented as colored circles. Proteins associated with each snRNA are highlighted in color. For details, see the text.

Figure 3. Assembly and dissociation cycle of the spliceosome. The stepwise interaction of the spliceosomal snRNPs (colored circles) during the removal of an intron from a pre‐mRNA containing two exons is depicted. Only the spliceosomal complexes that can be resolved biochemically in splicing extracts are shown. Eight evolutionarily conserved DExD/H‐type RNA‐dependent ATPases/helicases act at specific steps of the splicing cycle to catalyze RNA–RNA rearrangements and RNP‐remodeling events. These enzymes include Sub2 (UAP56 in humans), Prp5, Prp28, Brr2, Prp2, Prp16, Prp22, and Prp43 (with Brr2 and Prp22 acting at more than one step in the cycle). The GTPase Snu114 also functions at several steps during the cycle. In yeast, Prp28 acts at a later stage during spliceosome activation (the B complex to B act complex transition) [ 15]. Prp2 acts during the B act to B* complex transition [ 35]. Several of the other ATPases, such as Prp5, Prp16, and Prp22, carry out proofreading functions at the stages indicated.

Figure 4. Spliceosomal RNA network and molecular interactions at the 5′ splice site (5′SS), branch site (BS), and 3′ splice site (3′SS) within the spliceosomal B and B* complexes and the protein network within the spliceosomal E and A complexes.
  1. The network of RNA interactions in the pre‐catalytic (left) and catalytically activated (right) spliceosome. During activation of the spliceosome, regions of U6 and U2 (red and green) undergo major rearrangements. The 5′ end of the U6 snRNA base‐pairs through its highly conserved ACAGA motif to the 5′SS, displacing U1. U4 and U1 are destabilized or dissociate from the spliceosome at the time of activation and are no longer part of the spliceosome's RNA interaction network. Spliceosomal snRNAs are depicted with schematic secondary structures and are not drawn to scale. Only stem–loop I of U5 is shown. Critical base‐pairing interactions observed in yeast are highlighted;
  2. Top: In the spliceosomal E complex, the pre‐mRNA (exons, gray; introns, black) 5′SS is bound by the U1 snRNP and the BS by SF1/BBP, whereas the polypyrimidine tract and 3′SS are bound by the U2 auxiliary factor (U2AF) subunits U2AF 65 and U2AF 35, respectively. U2AF 65 binds both SF1/BBP and U2AF 35.
Intron bridging interactions occur also between SR proteins and RS domain‐containing subunits of U1 snRNP and U2AF. Bottom: Upon stable U2 snRNP binding during A‐complex formation, SF1/BBP is displaced, allowing the U2‐associated protein p14 to contact the BS and U2AF65 to interact with SF3b155. The U2/BS base‐pairing interaction is stabilized by components of the U2 snRNP and by the arginine‐serine‐rich (RS) domain of U2AF65.

Figure 5. Model for the catalytic activation of the spliceosome by Prp2 before the Cwc25‐promoted step 1 and subsequent catalysis of step 2.
  1. Network of RNA interactions in the activated (left column, B act) and catalytically activated (right column, B*) spliceosome. During catalytic activation of the spliceosome the U2 SF3a/b proteins are destabilized by Prp2/ATP, such that the BS adenosine becomes available for a nucleophile attack at the 5′SS (red arrow);
  2. The formation of a step 1 spliceosome (C complex) is then promoted by the heat‐stable protein Cwc25.
Subsequently, the step 1 spliceosome catalyzes step 2 in the presence of the RNA helicase Prp16, ATP and the step 2 splicing factors Slu7, Prp18 and Prp22. Critical base‐pairing interactions are highlighted in color; (c) Summary of the transition from an activated (B act) to a catalytically activated (B*); and finally to a step 1 spliceosome, with proteins required during these transitions [ 35].

Figure 6. Compositional dynamics of yeast spliceosomes. The protein composition of the yeast B, B act, and C complexes was determined by mass spectrometry. Proteins (yeast nomenclature) are grouped according to snRNP association, function, presence in a stable heteromeric complex, or association with a particular spliceosomal complex, as indicated. The relative abundance of proteins is indicated by light (substoichiometric amounts) or dark (stoichiometric amounts) lettering, and is based on the relative number of peptides sequenced [ 18].

Figure 7. Evolutionarily conserved blueprint for yeast and human spliceosomes. Yeast: Proteins (yeast nomenclature) evolutionarily conserved between yeast and human, associated with purified yeast B, B act, and C complexes, are placed inside the rectangle. Proteins above the rectangle do not have a human counterpart. Human: Proteins (human nomenclature) evolutionarily conserved between yeast and human, associated with purified human A, B, and C complexes, are placed inside the rectangle. Proteins below the rectangle were found associated with purified human spliceosomal complexes, but the majority of them do not have a yeast counterpart [ 34, 41]. Numbers indicate the total number of individual proteins in a particular group. Asterisks: proteins that do have homologs in yeast or human but were not found, or were found only very loosely, associated with purified spliceosomal complexes; for example, yeast Msl5, Npl3, Mud2 and Hub1. Cus2, Prp28 and Sad1 were not detected by mass spectrometry, and are included only for completeness, as is human TIA‐1, which is the homolog of yeast Nam8 [ 18]. Proteins are grouped as described in the legend of Figure 6.