1. mRNA Multiple processing and localization steps are required for proper mRNA maturation following the synthesis of the primary transcript. At a minimum, these include capping, splicing, 3´-end cleavage, polyadenylation, and nuclear export. If each of these 5 processes are 90% efficient, only ~59% of mRNAs will be properly matured. What happens to the improperly processed transcripts? – Do they get sent to the cytoplasm for protein synthesis anyway? – Do they get degraded before they can make potentially defective proteins?

2. 1. The splicing and processing components as well as export factors are co- transcriptionally recruited to the nascent transcript. 2. The correct mRNA forms the core of an export-competent mRNP that is eventually exported and expressed in the cytoplasm. Nuclear mRNA

3. Post-transcriptional Processes II: Pre-mRNA Polyadenylation Most cytoplasmic mRNAs have a polyA tail (3’ end) of 50-250 Adenylates – a notable exception is histone mRNAs Added post-transcriptionally by an enzyme, polyA polymerase(s) Turns over (recycles) in cytoplasm

4. Functions of the PolyA Tail 1.Promotes mRNA stability - Deadenylation (shortening of the polyA tail) can trigger rapid degradation of the mRNA 2.Enhances translation - promotes recruitment by ribosomes - bound by a polyA-binding protein in the cytoplasm called PAB1 - synergistic stimulation with Cap!

5. Polyadenylation (PolyA) Signals AAUAAA in mammals and plants Located ~20-30 bp from the polyA site – Other hexamers less efficient but are used Mutagenesis and in vivo expression studies reveal 2 other motifs downstream of AAUAAA that are important: 1.GU-rich stretch 2.U-rich stretch

6. Polyadenylation (PolyA) Signals AAUAAA in mammals and plants Located ~20-30 bp from the polyA site – Other hexamers less efficient but are used Mutagenesis and in vivo expression studies reveal 2 other motifs downstream of AAUAAA that are important: 1.GU-rich stretch 2.U-rich stretch

7. Polyadenylation: Mechanism Occurs in 2 phases – Phase 1: requires AAUAAA and ~8 nt downstream (3’) – Phase 2 : Once ~10 As are added, further adenylation does not require the AAUAAA

8. Proteins Required for Polyadenylation Phase I: 1.CPSF 2.PolyA polymerase Phase II: 1.PolyA polymerase (PAP II) 2.PolyA Binding Protein II (PAB II) - PAB II binds to short A-tail - Helps PAP synthesize long tails

9. Cleavage and polyadenylation of pre-mRNAs in mammalian cells 1.Cleavage-and-polyadenylation specificity factor (CPSF) binds to an upstream AAUAAA polyadenylation signal. 2.CSTF interacts with a downstream GU- or U- rich sequence and with bound CPSF, forming a loop in the RNA; binding of CFI and CFII help stabilize the complex. 3.Binding of poly(A) polymerase (PAP) then stimulates cleavage at a poly(A) site, which usually is 10 – 35 nucleotides 3' of the upstream polyadenylation signal. 4.The cleavage factors are released, as is the downstream RNA cleavage product, which is rapidly degraded. Bound PAP then adds ≈12 A residues at a slow rate to the 3'-hydroxyl group generated by the cleavage reaction. 5.Binding of poly(A)-binding protein II (PABII) to the initial short poly(A) tail accelerates the rate of addition by PAP. After 200 – 250 A residues have been added, PABII signals PAP to stop polymerization.