- poly(A)+ mRNA is mRNA that has a 3 terminal stretch of poly(A).
- Poly(A) polymerase is the enzyme that adds the stretch of polyadenylic acid to the 3 of eukaryotic mRNA. It does not use a template.
- Poly(A)-binding protein (PABP) is the protein that binds to the 3 stretch of poly(A) on a eukaryotic mRNA.
- cDNA is a single-stranded DNA complementary to an RNA, synthesized from it by reverse transcription in vitro.
- poly(A)- mRNA is mRNA that has does not have a 3 terminal stretch of poly(A).
- A length of poly(A) ~200 nucleotides long is added to a nuclear transcript after transcription.
- The poly(A) is bound by a specific protein (PABP).
- The poly(A) stabilizes the mRNA against degradation.
The 3 terminal stretch of A residues is often described as the poly(A) tail; and mRNA with this feature is denoted poly(A)+.
The poly(A) sequence is not coded in the DNA, but is added to the RNA in the nucleus after transcription. The addition of poly(A) is catalyzed by the enzyme poly(A) polymerase, which adds ~200 A residues to the free 3-OH end of the mRNA. The poly(A) tract of both nuclear RNA and mRNA is associated with a protein, the poly(A)-binding protein (PABP). Related forms of this protein are found in many eukaryotes. One PABP monomer of ~70 kD is bound every 10-20 bases of the poly(A) tail. So a common feature in many or most eukaryotes is that the 3 end of the mRNA consists of a stretch of poly(A) bound to a large mass of protein. Addition of poly(A) occurs as part of a reaction in which the 3 end of the mRNA is generated and modified by a complex of enzymes (see 24.19 The 3 ends of mRNAs are generated by cleavage and polyadenylation) (Darnell et al., 1971).
Binding of the PABP to the initiation factor eIF4G generates a closed loop, in which the 5 and 3 ends of the mRNA find themselves held in the same protein complex (see Figure 6.20 in 6.9 Eukaryotes use a complex of many initiation factors). The formation of this complex may be responsible for some of the effects of poly(A) on the properties of mRNA. Poly(A) usually stabilizes mRNA. The ability of the poly(A) to protect mRNA against degradation requires binding of the PABP.
Removal of poly(A) inhibits the initiation of translation in vitro, and depletion of PABP has the same effect in yeast in vivo. These effects could depend on the binding of PABP to the initiation complex at the 5 end of mRNA. There are many examples in early embryonic development where polyadenylation of a particular mRNA is correlated with its translation. In some cases, mRNAs are stored in a nonpolyadenylated form, and poly(A) is added when their translation is required; in other cases, poly(A)+ mRNAs are de-adenylated, and their translation is reduced.
The presence of poly(A) has an important practical consequence. The poly(A) region of mRNA can base pair with oligo(U) or oligo(dT); and this reaction can be used to isolate poly(A)+ mRNA. The most convenient technique is to immobilize the oligo(U or dT) on a solid support material. Then when an RNA population is applied to the column, as illustrated in Figure 5.19, only the poly(A)+ RNA is retained. It can be retrieved by treating the column with a solution that breaks the bonding to release the RNA.
The only drawback to this procedure is that it isolates all the RNA that contains poly(A). If RNA of the whole cell is used, for example, both nuclear and cytoplasmic poly(A)+ RNA will be retained. If preparations of polysomes are used (a common procedure), most of the isolated poly(A)+ RNA will be active mRNA. However, in addition to mRNA in polysomes, there are also ribonucleoprotein particles in the cytosol that contain poly(A)+ mRNA, but which are not translated. This RNA may be "stored" for use at some other time. Isolation of total poly(A)+ mRNA therefore does not correspond exactly with the active mRNA population.
The "cloning" approach for purifying mRNA uses a procedure in which the mRNA is copied to make a complementary DNA strand (known as cDNA). Then the cDNA can be used as a template to synthesize a DNA strand that is identical with the original mRNA sequence. The product of these reactions is a double-stranded DNA corresponding to the sequence of the mRNA. This DNA can be reproduced in large amounts.
The availability of a cloned DNA makes it easy to isolate the corresponding mRNA by hybridization techniques. Even mRNAs that are present in only very few copies per cell can be isolated by this approach. Indeed, only mRNAs that are present in relatively large amounts can be isolated directly without using a cloning step.
Almost all cellular mRNAs possess poly(A). A significant exception is provided by the mRNAs that code for the histone proteins (a major structural component of chromosomal material). These mRNAs comprise most or all of the poly(A)- fraction. The significance of the absence of poly(A) from histone mRNAs is not clear, and there is no particular aspect of their function for which this appears to be necessary (for review see Jackson and Standart, 1990).