Lecture 16: Cleavage and Polyadenylation

Polyadenylation

After cleavage of the mRNA, a slow phase of polyadenylation occurs in which 12 A residues are added to the 3' OH. Followed by a fast phase of polyadenylation where up to 250 more A residues are added by PAP.

In vitro Experiments Examining Polyadenylation

1. cDNA template + RNA polymerase generates mRNA in vitro. 2. mRNA + PAP generates polyadenylated mRNA.

Cleavage and Polyadenylation Specificity Factor (CPSF)

A large 4-subunit protein (~360 kDa) that an unstable complex with the the AAUAAA sequence that is subsequently stabilized by the addition of at least 4 separate protein complexes that bind to the CPSF-RNA complex. Bound to the C-terminal domain of RNA polymerase.

Alternative Cleavage and Polyadenylation (APA)

Associated with cancer cells. Cancer cell lines express shorter RNAs with shorter 3' UTRs.

Transcription Termination

At the end of transcription the RNA is cleaved by an endonuclease and a poly(A) tail is added to the exposed 3' end. The release of RNAPII from the DNA template occurs at diffuse positions hundreds of bases downstream of the poly(A) signal and termination requires a functional poly(A) site. The RNAPII CTD provides a platform for holding RNA binding proteins that recognize the poly(A) signal (CPSF, CstF). Poly(A) tail is not encoded in the RNA but the poly(a) signal sequence is.

snRNP U7

Binds to HDE.

SLBP

Binds to the stem loop sequence.

PABPII

Contains an RRM motif. Binds to the short A tail initially added and then stimulates further polymerization of residues by PAP. May also signal to PAP to terminate addition of A residues after 250 are added.

Role of 3'UTR

Different 3' UTR isoforms can allow for differential control of gene expression. Shorter 3' UTR lead to cancer. Controls how much a gene is translated. Contains alternative cleavage and polyadenylation sequences. Contains miRNA binding sequences which help break down the mRNA and bring the mRNA into the P bodies where they are slowly degraded.

HDE (Histone Downstream Element)

Functions in 3' processing through base pairing to a complementary sequence in the snRNA of the U7 snRNP. Sequence: CAAGAAAGA. Very highly conserved.

CF1/2 (Cleavage Factor 1 and 2)

Help to stabilize the CPSF-RNA complex.

CstF (Cleavage Stimulatory Factor)

Interacts with G/U rich sequence to stabilize the CPSF-RNA complex. Bound to the C-terminal domain of RNA polymerase.

Histone Genes

Mature 3' end of histones is formed by endonucleolytic cleavage of the primary transcript. Endonucleolytic cleavage occurs downstream of the stem-loop. Cleavage site is determined by U7 snRNP:HDE base pairing. RNAPII termination does not occur until several hundred base pairs downstream of 3' cleavage site. 3' end of mammalian mature histone mRNA contains conserved ~28 nucleotide sequence encompassing a stem-loop structure up to 70 nucleotides downstream of the stop codon and is bound by SLBP. Multiple copies in the genome and the lack of introns because they are needed quickly.

snRNA Genes

No introns, not spliced nor polyadenylated.

PAP (Poly(A) Polymerase)

Type of RNA polymerase. Binds to complex before cleavage occurs to stabilize the CPSF-RNA complex. Ensures that the free 3' end of the cleaved mRNA is polyadenylated as quickly as possible. Adds up to 250 more A residues to the poly(A) tail. Doesn't require a template strand.

Sequences Required for Cleavage and Polyadenylation

mRNAs contain the signal sequence AAUAAA that is situated 10-15 nucleotides upstream of the poly(A) tail. A second downstream signal that is a G/U rich sequence that is required for efficient cleavage and polyadenylation and is located ~ 50 nucleotides from the site of cleavage.