Poly-T tail

Mature mRNA transcripts (sense strand) from eukaryotic cells can be purified and then reverse transcribed, with the assistance of a reverse transcriptase enzyme (from Moloney murine leukemia virus, MMLV), into complementary DNAs (cDNAs) that will anneal with the mRNA transcripts by Watson-Crick base pairing to give anti-parallel DNA/RNA duplexes or double helices. The poly(A) tail in each mature mRNA transcript is actually a usefiil handle for each reverse transcriptase reaction. Thereafter, DNA/RNA duplexes must be broken down with the assistance ofRNAse enzymes (specific for the hydrolysis of RNA phospho diester links) and a sense strand of DNA constructed instead on each cDNA single strand so that equivalent, more stable antiparallel DNA/DNA duplexes are generated instead, with the assistance of a DNA polymerase enzyme. In this instance, the poly(T) tail in each cDNA molecule turns out to be important for the DNA polymerase reaction ... 

The presence of the poly-A tail has been very fortuitous for researchers. By designing an affinity chromatography column (Chapters 5 and 13) with a poly-T tail (or poly[d(T)] tail), the isolation of mRNA from a cell lysate... 

The cDNA prepared from mRNA would have a long poly(T) tail, unlike genomic DNA. Remember that the poly(A) tail is added to the 3 end of mammalian mRNA and that there is no counterpart on DNA. A second striking difference would be that the cDNA would contain no intervening sequences (introns) and would therefore be much shorter... 

Due to the sequencing orientation of the original data, we found two major outputs available from SRA data poly(A) tail at 3 -end and poly(T) tail at the 5 -ends. These two situation are normal polyadenylation results. We suggest user to separate them into different processes, because A rich section and T rich section maybe appear at same sequence read. 

Bergamini, G., Preiss, T., and Hentze, M. W. (2000). Picomavirus IRESes and the poly(A) tail ioindy promote cap-independent translation in a mammalian cell-free system. RNA 6, 1781-1790. 

Humphreys, D. T., Westman, B. J., Martin, D. I., andPreiss, T. (2005). MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc. Natl. Acad. Sci. USA 102, 16961-16966. 

Woolstencroft, R. N., Beilharz, T. H., Cook, M. A., Preiss, T., Durocher, D., and Tyers, M. (2006). Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length. J. Cell Sci. 119, 5178-5192. 

Hoshino S, Imai M, Kobayashi T, Uchida N, Katada T (1999) The eukaryotic pol>peptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3 -poly(A) tail of mRNA. J Biol Chem 274 16677-16680... 

Whitley, P., Grahn, E., Kutay, U., Rapoport, T., and von Heijne, G. (1996). A12 residues long poly-leucine tail is sufficient to anchor synaptobrevin to the ER membrane. J. Biol. Chem. 271, 7583-7586. 

The mRNAs coding for nearly all mammalian proteins contain a tail of adenylate molecules. This tail is called poly (A). The tail occurs only at one end of the mRNA molecule. Conversion of the mRNA to the mRMA/DNA hybrid takes advantage of poly (A). This conversion is catalyzed by reverse transcriptase. Reverse transcriptase cannot bind to mRNA as is. It requires a primer for activation. The primer can take the form of a short strand of poly(dT). 1 he polyfdT) primer binds to the poly(A) tail of the mRNA, forming a small hybridized region. Formation of the hybrid is possible because A binds to T. 

Fig. 14.4. A schematic view of a eukaryotic gene, and steps required to produce a protein product. The gene consists of promoter and transcribed regions. The transcribed region contains introns, which do not contain coding sequence for proteins, and exons, which do carry the coding sequences for proteins. The first RNA form produced is heterogenous nuclear RNA (hn RNA), which contains both intronic and exonic sequences. The hnRNA is modified such that a cap is added at the 5 end (cap site), and a poly-A tail added to the 3 end. The introns are removed (a process called splicing) to produce the mature mRNA, which leaves the nucleus to direct protein synthesis in the cytoplasm. Py is pyrimidine (C or T).

Poly(vinyl alcohol) is very high in head to tail strucmres, based on NMR data. It shows the presence of only a small amount of adjacent hydroxyl groups. The polymer prepared from amorphous poly(vinyl acetate) is crystalline, because the relatively small size of the hydroxyl groups permits the chains to line-up into crystalline domains. Synthesis of isotactic poly(vinyl alcohol) was reported from isotactic poly(vinyl ethers), like poly(benzyl vinyl ether), poly(t-butyl vinyl ether), poly (trimethylsilyl vinyl ether), and some divinyl compounds. 

Figure 2 shows some possible cases of paired-end sequences. If PAT2 is with a poly(A) tail (type PAT2 A), the oligo(A) of the sequence in PAT2 is trimmed, so as the oligo(T) of the PATI trimmed. Both sequences are mapped to the same locus, and the cleavage site can be defined as marked (Fig. 2a). If PAT2 is without a poly(A) tail (type PAT2 N), then PAT2 and PATI will be mapped to different loci but within a certain distance (Fig. 2b).

One of the key experimental procedures is the systematic removal of rRNA from the mRNA fraction. Typically, mRNA has a long poly A tail that can be captured on a poly T chromatography column or batch resin. The rRNA and tRNAs pass through the column and, after a suitable washing protocol, the mRNA fraction is concentrated and stored for subsequent experimentation. The effectiveness of the purification of a typical mRNA purification scheme was followed by RNA chromatography by Hornby and coworkers [34]. ft was found that at least two rounds of enrichment are usually required in order to remove the bulk of the nonpoly A RNA. 

FRA Franco, T.T., Galaev, I.Yu., Hatti-Kaul, R., Holmbeig, N., Biilow, L., and Mattiasson, B., Aqueous two-phase system formed by thermoieactive viityl imidazoleA inyl eaprolactam copolymer and dextran for partitioning of a protein with a poly histidine tail, Biotechnol. Techn., 11, 231, 1997. 

Preiss, T., M. Muckenthaler, and M.W. Hentze, Poly(A)-tail-promoted translation in yeast implications for translational control. Rna, 1998. 4(11) p. 1321-31. 

Preiss, T. and M.W. Hentze, Dual function of the messenger RNA cap structure in poly(A)-tail- promoted translation in yeast. Nature, 1998. 392(6675) p. 516-20. 

S. Amou, O. Haba, K. Shirato, T. Hayakawa, M. Ueda, K. Takeuchi, and M. Asai, Head-to-tail regioregularity of poly(3-hexylthiophene) in oxidative coupling polymerization with FeCl3, J. Polym. Sci., Part A Polym. Chem., 37 1943-1948, 1999. 

T.A. Chen and R.D. Rieke, The first regioregular head-to-tail poly(3-hexylthiophene-2,5-diyl) and a regiorandom isopolymer nickel versus palladium catalysis of 2(5)-bromo-5(2)- (bromozincio)-3-hexylthiophene polymerization, J. Am. Chem. Soc., 114 10087-10088, 1992. 

Properties of PIB are shown in Table 6.4. Unsymmetric monomers may form polymers that are in the form of head to tail (H-T) and head to head (H-H) arrangements. The H-T arrangement is most common. Mostly these variations in the arrangements are not important. However, as a specific example, it has been shown that in poly(vinyl chloride) the H-H and the H-T species are immiscible, whereas for PIB the H-H and the H-T species are miscible over a wide range of compositions (19). 

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