3'-end Cleavage and Polyadenylation.

Virtually all mRNAs have a number of adenylate residues at their 3'-end; the poly(A)-tail. These A's are not encoded in the genes. Instead, the pre-mRNA is cleaved at a defined site and a poly(A) polymerase adds 150-200 adenylate residues. The most important function of the poly(A)-tail is during translation. The length of the poly(A)-tail can be regulated in the cytoplasm. In some species, for example, the egg cell stores mRNA in the cytoplasm for later use after fertilization. The stored mRNA has a short poly(A)-tail. Activation of the mRNA for translation includes lengthening of the poly(A)-tail. It has also been suggested that the poly(A)-tail is involved in determining how long an mRNA is present in the cell before it is degraded.

3'-cleavage and polyadenylation are closely coupled to the termination of transcription. The RNA polymerase transcribes the DNA template several hundreds of nucleotides downstream of the cleavage and polyadenylation site. Specific sequence signals in the pre-mRNA direct the binding of protein factors to the pre-mRNA. A conserved AAUAAA sequence is absolutely necessary and there are also less well-conserved sequences on both sides of the AAUAAA sequence, e.g. a U-rich sequence downstream, called DSE (Downstream Element). Some of these factors are believed to bind to RNA polymerase II and travel along during transcription. Based on biochemical experiments, a scheme for the cleavage and polyadenylation has been worked out. In this scheme, several protein factors are identified, such as CPSF (Cleavage and Polyadenylation Specificty Factor), CstF (Cleavage stimulation Factor) and CF I and CF II (Cleavage Factors I and II). A special feature of the polyadenylation reaction is that the poly(A) polymerase sticks poorly to the end of the pre-mRNA until approximately 20 A's have been added. Then, poly(A)-binding protein (PAB) has bound to the short poly(A)-tail and the poly(A) polymerase is more firmly bound until 150-200 A's are rapidly added. At this stage the poly(A)polymerase again tends to fall off.

The 3'-cleavage and the initial part of the polyadenylation reaction take place at the active gene and polyadenylation is presumably completed as the pre-mRNA-protein complex is transported away from the gene.

Polydenylation of pre-mRNA can be regulated in the cell in at least three different ways. First, there are alternative cleavage and polyadenylation sites for some genes. One example is during the development of B-lymphocytes. During this development, the synthesis of antibody molecules switches from membrane bound to secreted antibodies. The difference is due to a switch from one polyadenylation site to another. This results in a shorter antibody molecule, lacking a hydrophobic membrane-spanning part at the very end of the antibody molecule. Second, the very polyadenylation process can be modified. In one case, it has been shown that the protein product of an mRNA can bind to its own pre-mRNA and then inhibit polyadenylation. This results in degradation of the pre-mRNA and less production of the corresponding protein. Third, the expression of other genes can be regulated by changing the length of the poly(A)-tail in the cytoplasm. In some species, the oocyte stores mRNA in the cytoplasm for later use during the first cleavages after fertilization. These stored mRNAs have had their poly(A)-tail shortened in the cytoplasm. As the mRNAs are re-activated, their poly(A)-tails are extended by a cytoplasmic poly (A) polymerase.