Understand the structure of an intron in terms of the 5' and 3' splicing sites as well as the branch site (note: you do NOT need to know the specific consensus sequences except for the GU-AG rule and the invariant A)

Understand the theory behind the pathways in which introns are spliced out (referring to the order they are spliced out - see slide 8)

Describe the spliceosome complex and the snRNPs and snRNAs that are found within it

Describe the functional order of the snRNPs in intron splicing

Understand what a lariat loop is, when it is produced and what it is produced from

Understand the types of interactions that occur during intron splicing (RNA-RNA, RNA-protein, protein-protein)

Understand the role of U2AF and BBF in splicing

For the snRNAs, understand how and where they interact with their substrate. NOTE: you do not need to know the specific sequences of the base pairing

For the snRNPs, understand how they interact with each other and what their roles are

Understand why the amount of base pairing is important between the snRNAs and the regions of the introns

For the following mechanisms, understand/describe (to the detail discussed in lecture):

• The sequence features required for the mechanism to take place unless I have told you that you do not need to know them (for example, I want you to know that there is a 5' splice site with a consensus sequence, but you do not need to know what the consensus sequence is except for the GU)
• The steps of the mechanism
• The proteins involved at each step
• The outcome(s) of the mechanism

Mechanisms:

• Intron splicing
• Transesterification step 1
• Transesterification step 2
• In a general sense - splicing of group I introns
• In a general sense - splicing of group II introns

For mechanisms as well as the other proteins mentioned in the learning objectives, think about what would happen if you removed their functionality through mutagenesis.

Assays/techniques to be familiar with:

• Mutational analysis including compensatory mutations