Poly tail, mRNA

The first step in preparing a cDNA library Is to Isolate the total mRNA from the cell type or tissue of Interest. Because of their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs present In a cell extract by use of a column to which short strings of thymidyl-ate (oligo-dTs) are linked to the matrix. 

Recent studies with mRNAs stored in Xenopus oocytes have helped elucidate the mechanism of this type of translational control. Experiments in which short-tailed mRNAs are injected into oocytes have shown that two sequences in their 3 UTR are required for their polyadenylation in the cytoplasm the AAUAAA poly(A) signal that is also required for the nuclear polyadenylation of pre-mRNAs, and one or more copies of an upstream U-rich cytoplasmic polyadenyla... 

Prepare first-stranded target cDNA (see Note 17) from poly (A)+ mRNA as described above (preferred) or in vitro sense RNA derived from a target cDNA library. In the latter case one should use 5 pg of random hexamers as primers, since one cannot be sure that the poly A tail is present after Xhol digestion. Remove the RNA by adding 10 pL of 0.25 M EDTA and 30 pL 0.15 N NaOH, and incubating for 60 min at 65°C. Neutrahze with 30 pL of 0.15 N HCl, ethanol-precipitate, rinse, and dry. 

The cDNAs synthesized from poly(A)-tailed mRNA in the presence of the oligo(dT) primer range widely in size from 200 bases to several kilobases, all depending on reaction conditions. Reverse transcriptases lacking RNase H activities produce longer cDNAs and at substantially higher yields. 

Note that some poly(A) mRNAs, e.g., histone mRNA of yeast, do not carry the AAUAAA sequence in their 3 ends. In these mRNAs, two different classes of polyadenylation sites have been recognized, i.e., a unidirectional site with the sequence UUUUUAU and a bidirectional site with a tripartite sequence UAG UA(U)GU UUU (26). For other mRNAs that have no apparent recognition sequence motifs in their 3 UTR and yet carry poly (A) tails, other additional or alternative structural features may be present such as further internal sequences (5 to the apparent polyadenylation signal) or secondary structures that are required for proper and/or differential polyadenylation. 

Small nuclear RNAs (snRNAs), a subset of these RNAs, are significantly involved in mRNA processing and gene regulation. Of the several snRNAs, Ul, U2, U4, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA (Chapter 37). The U7 snRNA may be involved in production of the correct 3 ends of histone mRNA—which lacks a poly(A) tail. The U4 and U6 snRNAs may also be required for poly(A) processing. 

The relationship between hnRNA and the corresponding mature mRNA in eukaryotic cells is now apparent. The hnRNA molecules are the primary transcripts plus their early processed products, which, after the addition of caps and poly(A) tails and removal of the portion corresponding to the introns, are transported to the cytoplasm as mature mRNA molecules. 

As mentioned above, mammahan mRNA molecules contain a 7-methylguanosine cap structure at their 5 terminal, and most have a poly(A) tail at the 3 terminal. The cap stmcmre is added to the 5 end of the newly transcribed mRNA precursor in the nucleus prior to transport of the mELNA molecule to the cytoplasm. The S cap of the RNA transcript is required both for efficient translation initiation and protection of the S end of mRNA from attack by S —> S exonucleases. The secondary methylations of mRNA molecules, those on the 2 -hydroxy and the N of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm. 

Poly(A) tails are added to the S end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues. The poly(A) tail appears to protect the S end of mRNA from S —> S exonuclease attack. The presence or absence of the poly(A) tail does not determine whether a precursor molecule in the nucleus appears in the cytoplasm, because all poly(A)-tailed hnRNA molecules do not contribute to cytoplasmic mRNA, nor do all cytoplasmic mRNA molecules contain poly(A) tails... 

Biochemical and genetic experiments in yeast have revealed that the b poly(A) tail and its binding protein, Pablp, are required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. Pablp, bound to the poly(A) tail, interacts with eIF-4G, which in turn binds to eIF-4E that is bound to the cap structure. It is possible that a circular structure is formed and that this helps direct the 40S ribosomal subunit to the b end of the mRNA. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. It appears that a similar mechanism is at work in mammalian cells. 

Figure 39-19. Structure of a typical eukaryotic mRNA showing elements that are involved in regulating mRNA stability. The typical eukaryotic mRNA has a 5 noncoding sequence (5 NCS), a coding region, and a 3 NCS. All are capped at the 5 end, and most have a polyadenylate sequence at the 3 end. The 5 cap and 3 poly(A) tail protect the mRNA against exonuclease attack. Stem-loop structures in the 5 and 3 NCS, features in the coding sequence, and the AU-rich region in the 3 NCS are thought to play roles in mRNA stability.

The advantage of mRNA over plasmid transfection is the ability of in vitro transcription to allow precise control over features contained within the mRNA (Humphreys et al., 2005 Pillai et al., 2005 Westman et al, 2005). For example, mRNA can be prepared either with or without the physiological m7G(5/)ppp(5/)G cap structure and S poly(A) tail, which are important mediators of canonical translation initiation (Gallie, 1991 Hentze et al., 2006 Iizuka et al, 1994 Kahvejian et al, 2005 Tarun and Sachs, 1995). 

At this stage, mRNA can be polyadenylated using the poly(A) tailing kit (Ambion), according to the manufacturer s instructions. 

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