11. Cleavage of the 3 end may require a small RNA

22.10 The 3′ ends of mRNAs are generated by cleavage

Key terms defined in this section

Cordycepin is 3´ deoxyadenosine, an inhibitor of polyadenylation of RNA.
Endonucleases cleave bonds within a nucleic acid chain; they may be specific for RNA or for single-stranded or double-stranded DNA.

It is not clear whether RNA polymerase II actually engages in a termination event at a specific site. It is possible that its termination is only loosely specified. In some transcription units, termination occurs >1000 bp downstream of the site corresponding to the mature 3′ end of the mRNA (which is generated by cleavage at a specific sequence). Instead of using specific terminator sequences, the enzyme ceases RNA synthesis within multiple sites located in rather long "terminator regions." The nature of the individual termination sites is not known.

The 3′ ends of mRNAs are generated by cleavage followed by polyadenylation. Addition of poly(A) to nuclear RNA can be prevented by the analog 3′-deoxyadenosine, also known as cordycepin. Although cordycepin does not stop the transcription of nuclear RNA, its addition prevents the appearance of mRNA in the cytoplasm. This shows that polyadenylation is necessary for the maturation of mRNA from nuclear RNA.

Figure 22.30 The sequence AAUAAA is necessary for cleavage to generate a 3 F end for polyadenylation.

Generation of the 3′ end is illustrated in Figure 22.30. RNA polymerase transcribes past the site corresponding to the 3′ end, and sequences in the RNA are recognized as targets for an endonucleolytic cut followed by polyadenylation.

A common feature of mRNAs in higher eukaryotes (but not in yeast) is the presence of the sequence AAUAAA in the region from 11 V30 nucleotides upstream of the site of poly(A) addition. The sequence is highly conserved; only occasionally is even a single base different. Deletion or mutation of the AAUAAA hexamer prevents generation of the polyadenylated 3′ end. The signal is needed for both cleavage and polyadenylation (Conway and Wickens, 1985; Gil and Proudfoot, 1987; for review see Wahle and Keller, 1992).

Figure 22.31 The 3 F processing complex consists of several activities. CPSF and CstF each consist of several subunits; the other components are monomeric. The total mass is >900 kD.

The development of a system in which polyadenylation occurs in vitro opened the route to analyzing the reactions. The formation and functions of the complex that undertakes 3′ processing are illustrated in Figure 22.31. Generation of the proper 3′ terminal structure requires an endonuclease (consisting of the components CFI and CFII) to cleave the RNA, a poly(A) polymerase (PAP) to synthesize the poly(A) tail, and a specificity component (CPSF) that recognizes the AAUAAA sequence and directs the other activities. A stimulatory factor, CstF, binds to a G-U-rich sequence that is downstream from the cleavage site itself (Takagaki et al., 1988).

The specificity factor contains 4 subunits, which together bind specifically to RNA containing the sequence AAUAAA. The individual subunits are proteins that have common RNA-binding motifs, but which by themselves bind nonspecifically to RNA. Protein-protein interactions between the subunits may be needed to generate the specific AAUAAA-binding site. CPSF binds strongly to AAUAAA only when CstF is also present to bind to the G-U-rich site.

The specificity factor is needed for both the cleavage and polyadenylation reactions. It exists in a complex with the endonuclease and poly(A) polymerase, and this complex usually undertakes cleavage followed by polyadenylation in a tightly coupled manner.

The two components CFI and CFII (cleavage factors I and II), together with specificity factor, are necessary and sufficient for the endonucleolytic cleavage.

The poly(A) polymerase has a nonspecific catalytic activity. When it is combined with the other components, the synthetic reaction becomes specific for RNA containing the sequence AAUAAA. The polyadenylation reaction passes through two stages. First, a rather short oligo(A) sequence (~10 residues) is added to the 3′ end. This reaction is absolutely dependent on the AAUAAA sequence, and poly(A) polymerase performs it under the direction of the specificity factor. In the second phase, the oligo(A) tail is extended to the full ~200 residue length. This reaction requires another stimulatory factor that recognizes the oligo(A) tail and directs poly(A) polymerase specifically to extend the 3′ end of a poly(A) sequence.

The poly(A) polymerase by itself adds A residues individually to the 3′ position. Its intrinsic mode of action is distributive; it dissociates after each nucleotide has been added. However, in the presence of CPSF and PABP (poly(A)-binding protein), it functions processively to extend an individual poly(A) chain. The PABP is a 33 kD protein that binds stoichiometrically to the poly(A) stretch. The length of poly(A) is controlled by the PABP, which in some way limits the action of poly(A) polymerase to ~200 additions of A residues. The limit may represent the accumulation of a critical mass of PABP on the poly(A) chain. PABP remains a component of mRNA; its exact role in mRNA metabolism has yet to be defined.

Some mRNAs are not polyadenylated. The formation of their 3′ ends is therefore different from the coordinated cleavage/polyadenylation reaction. The most prominent members of this mRNA class are the mRNAs coding for histones. Formation of their 3′ ends depends upon secondary structure. The RNA terminates in a stem-loop structure, and mutations that prevent formation of the duplex stem prevent formation of the end of the RNA. Secondary mutations that restore duplex structure (though not necessarily the original sequence) behave as revertants. This suggests that formation of the secondary structure is more important than the exact sequence.

Either or both of the DNA strands could in principle be involved in forming secondary structure. They can be distinguished by using templates consisting of heteroduplex molecules, in which the two strands of DNA are not identical. It turns out that it is important to be able to write a duplex structure for the coding strand, not the strand used as template. This suggests that the secondary structure exerts its effect by forming in the RNA as it is transcribed.

Reviews

Wahle, E. and Keller, W. (1992). The biochemistry of 3?/FONT>-end cleavage and polyadenylation of messenger RNA precursors. Ann. Rev. Biochem 61, 419-440.

Research

Conway, L. and Wickens, M. (1985). A sequence downstream of AAUAAA is required for formation of SV40 late mRNA 3?/FONT> termini in frog oocytes. Proc. Nat. Acad. Sci. USA 82, 3949-3953.

Gil, A. and Proudfoot, N. (1987). Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit &#szlig;-globin mRNA 3?/FONT> end formation. Cell 49, 399-406.

Takagaki, Y., Ryner, L. C., and Manley, J. L. (1988). Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell 52, 731-742.