Polyadenylate tail

Eukaryotic mRNAs often have long 3 untranslated sequences— sequences that follow the stop codon for the protein they encode. These mRNAs generally conclude with a sequence of up to 200 adenosines, the polyadenylic acid (polyA) sequence at the 3 end. This sequence isn t coded by the DNA template for the gene it is added post-transcriptionally. Not all mRNAs are polyadenylated. For example, histone mRNAs lack polyA tails. Polyadenylation seems to play a role in regulating the stability of mRNAs. An early event in the breakdown of some mRNAs is the removal of their polyA tails. 

After the gene is transcribed (i.e., posttranscription), regulation can occur during processing of the RNA transcript (hnRNA) into the mature mRNA. The use of alternative splice sites or sites for addition of the poly(A) tail (polyadenylation sites) can result in the production of different mRNAs from a single hnRNA and, consequently, in the production of different proteins from a single gene. 

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.

Fig. 1 Schematic outline of procedures employed in the synthesis of a cDNA gene copy from a polyadenylated mRNA template, insertion of the cDNA into a bacterial plasmid vector by a homopolymer tailing strategy, and cloning of the recombinant plasmid in an Escherichia coli host.

Figure 1. Expression of c-mos in mouse oocytes, c-mos is transcribed during oocyte growth and transcripts with short poly(A) tails are accumulated in fully-grown germinal vesicle (GV) stage oocytes. These transcripts are polyadenylated and translated following the resumption of meiosis and then degraded following fertilization and cleavage to the two-cell stage.

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

Example experiments using the previous methodologies are shown in Fig. 6.1. The major mRNA constructs described in this chapter are dia-grammatically represented in Fig. 6. IB and an example of in vitro transcribed and polyadenylated R-luc-4 sites mRNA is shown in Fig. 6.1A. In these experiments, translation of R-luc-4 sites mRNA is synergistically promoted by the physiological cap structure and the poly (A) tail (Fig. 6.1C), and full miR-dependent translational repression requires the presence of both modifications (Fig. 6.ID, Humphreys etal., 2005). (TheEMCV IRES-containing constructs are discussed later.)... 

PolyA Signal This is the polyadenylation signal for attachment of the polyA tail to generate mature mRNA it is important for transcription termination. 

Polyadenylation This process is the addition of many AMP molecules to the 3 end of the RNA molecule. Once bound it is known as a poly A tail, since it is comprised of 100 or more such adenylate (AMP) residues. The tail is thought to stabilise the mRNA molecule in the cytosol, probably by preventing hydrolysis by an RNAase. 

Fukaryotic mRNAs are modified by addition of a 7-methylguanosine residue at the 5 end and by cleavage and polyadenylation at the 3 end to form a long poly(A) tail. 

Addition of a poly-A tail Most eukaryotic mRNAs (with several notable exceptions, including those coding for the histones and some interferons) have a chain of 40 to 200 adenine nucleotides attached to the 3 -end (see Rgure 30.17). This poly-A tail is not transcribed from the DNA, but rather is added after transcription by the nuclear enzyme, polyadenylate polymerase. A consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3 -end of the RNA molecule, signals that a poly-A tail is to be added to the mRNA. These tails help stabilize the mRNAs and facilitate their exit from the nucleus. After the mRNA enters the cytosol, the poly-A tail is gradually shortened. 

A poly(A) "tail" consisting of -250 residues of adenylic acid is added next by poly(A) polymerase, a component of an enzyme complex that also cleaves the RNA chains.545 57111 Most eukaryotic mRNA is polyadenylated with the exception of that encoding histones. The function of the poly(A) is unclear. It is needed for transport of mRNA out of the nucleus, but it does confer a greatly increased stability to the mRNA in the cytoplasm where the adenylate irnits are gradually removed.307 308 In contrast, in chloroplasts and plant mitochondria polyadenylation is required for rapid degradation of mRNA.571c d Polyadenylation may also increase the efficiency of translation.572 Polyadenylation occurs rapidly within -1 min after transcription is completed. 

Most pre-mRNA transcripts are cleaved post-transcriptionally near the 3 end between a polyadenylation signal (5 -AAUAAA-3 ) and 5 -YA-3 (where Y = a pyrimidine). A GU-rich sequence may also be located further downstream. Specific proteins bind to these sequence elements to form a complex. One of the bound proteins, poly(A) polymerase, then adds a poly(A) tail of up to 250 A residues to the new 3 end of the RNA molecule and poly(A) binding protein molecules bind to this. The poly(A) tail protects the 3 end of the final mRNA against nuclease degradation and also increases translational efficiency of the mRNA. Some pre-mRNAs (e.g. histone pre-mRNAs) are cleaved near the 3 end but no poly(A) tail is added. 

The eukaryotic transcript undergoes post-transcriptional modifications such as capping (5 -methyl-GTP cap) and 3 -polyadenylation (3 -poly A tail). 

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