We have seen that the 5ʹ end of the pre-mRNA produced by RNA polymerase II is capped almost as soon as it emerges from the RNA polymerase. Then, as the polymerase continues its movement along a gene, the spliceosome assembles on the RNA and delineates the intron and exon boundaries. The long C-terminal tail of the RNA polymerase coordinates these processes by transferring capping and splicing components directly to the RNA as it emerges from the enzyme. In this section, we shall see that, as RNA polymerase II reaches the end of a gene, a similar mechanism ensures that the 3ʹ end of the pre-mRNA is appropriately processed.
The position of the 3ʹ end of each mRNA molecule is specified by signals encoded in the genome (Figure 6–34). These signals are transcribed into RNA as the RNA polymerase II moves through them, and they are then recognized (as RNA) by a series of RNA-binding proteins and RNA-processing enzymes (Figure 6–35). Two multisubunit proteins, called CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor), are of special importance.
intron sequences normal mRNA is formed from three exons
A SINGLE-NUCLEOTIDE CHANGE THAT DESTROYS A NORMAL SPLICE SITE, THEREBY CAUSING EXON SKIPPING mRNA with exon 2 missing
A SINGLE-NUCLEOTIDE CHANGE THAT DESTROYS A NORMAL SPLICE SITE, THEREBY ACTIVATING A CRYPTIC SPLICE SITE mRNA with extended exon 3 mRNA with extra exon inserted between exon 2 and exon 3
Figure 6–33 Abnormal processing of the β-globin primary RNA transcript in humans with the disease β thalassemia.
In the examples shown, the disease (a severe anemia due to aberrant hemoglobin synthesis) is caused by splice-site mutations found in the genomes of affected patients. The dark blue boxes represent the three normal exon sequences; the red lines connect the 5ʹ and 3ʹ splice sites that are used. In (B), (C), and (D), the light blue boxes depict new nucleotide sequences included in the final mRNA molecule as a result of the mutation denoted by the  black arrowhead. Note that when a mutation leaves a normal splice site without a partner, an exon is skipped (B) or one or more abnormal cryptic splice sites nearby is used as the partner site (C). [Adapted in part from S.H. Orkin, in The Molecular Basis of Blood Diseases (G. Stamatoyannopoulos et al., eds.), pp. 106–126. Philadelphia: Saunders, 1987.] degraded in the nucleus
Both of these proteins travel with the RNA polymerase tail and are transferred to the 3ʹ-end processing sequence on an RNA molecule as it emerges from the RNA polymerase.
Once CstF and CPSF bind to their recognition sequences on the emerging RNA molecule, additional proteins assemble with them to create the 3ʹend of the mRNA. First, the RNA is cleaved from the polymerase (see Figure 6–35). Next an enzyme called poly-A polymerase (PAP) adds, one at a time, approximately 200 A nucleotides to the 3ʹ end produced by the cleavage. The nucleotide precursor for these additions is ATP, and the same type of 5ʹ-to-3ʹ bonds are formed as in conventional RNA synthesis. But unlike other RNA polymerases, poly-A polymerase does not require a template; hence the poly-A tail of eukaryotic mRNAs is not directly encoded in the genome. As the poly-A tail is synthesized, proteins called poly-A-binding proteins assemble onto it and, by a poorly understood mechanism, help determine the final length of the tail.