3′-exonuclease, production and use thereof

Details 3′-exonuclease, production and use thereof 3′-exonuclease, production and use thereof

2000-12-15

2002-09-17

Prouty, Rebecca E. (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C435S199000, C530S412000

Reexamination Certificate

active

06451307

ABSTRACT:

DESCRIPTION
The present invention relates to a poly(A)-specific 3′-exonuclease activity which can be obtained by chromatographically purifying a crude extract of animal or human cells and to its use for deadenylating 3′-poly(A) tails of nucleic acids and as a pharmaceutical or diagnostic agent, or for identifying functional interactors.
Most eukaryotic mRNAs carry poly(A) tails of approx. 200 adenosine residues in length at their 3′ ends. These poly(A) tails appear to influence not only the half-life or intracellular transport of mRNAs but also translation of the mRNA into the corresponding protein. While the precise mechanism has still not been elucidated, the synthesis and degradation of poly(A) tails appear to be directly or indirectly connected with the function of the tails (see, e.g., Wickens, M. et al. (1977) Curr. Opin. Genet. Dev., 7, 220-232).
Poly(A)-degrading nuclease activities have already been investigated in several eukaryotic systems (see, e.g., Virtanen, A. & Åström, J. (1997) in Prog. Mol. Subcell. Biol. (Jeanteur, P., ed.) Vol. 16, 199-220, Springer-Verlag, Berlin-Heidelberg). Thus, two reaction pathways have, for example, been identified in yeasts. One of the reaction pathways, i.e. the so-called deadenylation dependent decapping passway, is started by removal of the poly(A) tail and concerns a 5′-3′-exonucleolytic degradation of mRNA by an Xm1p5′-exonuclease. The other reaction pathway, i.e. the so-called 3′-5′-decay passway, is started by a deadenylation of the mRNA and concerns a 3′-5′-exoribonucleolytic degradation of mRNA. A multicomponent complex, termed an exosome, has recently been identified as being involved in the 3′-5′-decay passway (Mitchell, P. et al. (1997), Cell 91, 457-466). The exosome consists of several 3′-5′-exoribonucleases and is involved both in 5.8S rRNA 3′ processing and in the 3′-5′ degradation of mRNA (Anderson, J. S. J. & Parker, R. (1998) EMBO J. 17, 1497-1506). However, it is not known whether any of the exoribonucleases of the exosome degrade poly(A) preferentially. In addition to this, a poly(A)-binding protein I (PABI)-dependent poly(A)-specific nuclease (PAN) has been identified in yeasts (see, e.g., Lowell, J. E. et al. (1992) Genes Dev. 6, 2088-2099). PAN is a 3′-5′-exoribonuclease and is composed of at least two polypeptides, i.e. Pan2p and Pan3p (see, e.g., Brown, C. E. Jr. et al. (1996) Mol. Cell. Biol., 16, 5744-5753).
At least three different poly(A)-degrading activities have been characterized in mammalian cells. For example, an activity found in Hela cells has a high selectivity for degrading 3′-located poly(A) tails, requires 3′-located hydroxyl groups and forms 5′-AMP as the mononucleotide reaction product (see, e.g., Åström, J. et al. (1992) J. Biol. Chem. 267 (25), 18154-18159 and J. Aström, A. Aström and A. Virtanen, In vitro deadenylation of mammalian mRNA by a HeLa cell 3′ exonuclease, EMBO J., (1991), Vol 10, 3067). Furthermore, an Mg
2+
-dependent poly(A)-specific 3′-exoribonuclease having a molecular weight of 74 kDa (Körner, C. G. & Wahle, E. (1997) J. Biol. Chem., 272 (16), 10448-10456) has been described in calf thymus. A polyribosome-associated 3′-exoribonuclease having a molecular weight of 33 kDa has also been described, but this 3′-exoribonuclease is not specific for poly(A) (Caruccio, N. & Ross, J. (1994) J. Biol. Chem. 269 (50), 31814-31821). Another poly(A)-specific 3′-exonuclease activity having a molecular weight of 60 kDa was identified in calf thymus. However, it subsequently turned out that this protein is the hnRNP L protein and consequently has no connection with the poly(A)-specific 3′-exoribonuclease activity which has been measured.
The object of the present invention was therefore to make available a 3′-exoribonuclease which specifically degrades 3′-located poly(A) tails.
The present invention therefore relates to a process for isolating a poly(A)-specific 3′-exonuclease activity, which process contains the following steps:
a) preparing a crude extract from animal or human cells;
b) precipitating the protein present in the crude extract obtainable from step (a);
c) subjecting the precipitate obtainable from step (b) to chromatography on a basic anion exchanger;
d) subjecting the active fractions from step (c) to affinity chromatography;
e) subjecting the active fractions from step (d) to chromatography on a basic anion exchanger;
f) subjecting the active fractions from step (e) to affinity chromatography;
g) subjecting the active fractions from step (f) to poly(A)-affinity chromatography;
h) subjecting the active fractions from step (g) to chromatography on a basic anion exchanger; and, where appropriate,
i) subjecting the active fractions from step (g) to gel filtration. Or
j) subjecting the active fractions from step (f) to two rounds of affinity chromatography;
Surprisingly, the 3′-exonuclease activity which can be obtained by the above-described process is specific for 3′-located poly(A) tails, with a 3′-located hydroxyl group being required and with 5′-AMP being formed as the mononucleotide reaction product. In contrast to the already known 3′-exonuclease activities, the 3′-exonuclease activity according to the invention has a molecular weight of approx. 50 kDa under denaturing conditions. In contrast to the 74 kDa protein from calf thymus, the 3′-exonuclease activity according to the invention is not stimulated by spermidine at low salt concentrations; on the contrary, if anything, it is inhibited both at low and at high salt concentrations. Furthermore, in contrast to the calf thymus 74 kDa protein, the 3′-exonuclease according to the invention interacts relatively strongly with Heparin Sepharose. Furthermore, the calf thymus 74 kDa protein is not found in the SDS-Page—
FIGS. 1B and 2B
. In addition, it was surprising that, in contrast to the HeLa cell exonuclease activity, the exonuclease according to the invention is also active in the presence of Mn
2+
. It is also surprising that the exonuclease according to the invention operates progressively, i.e. the exonuclease binds to the 3′ end of the poly(A) tail and degrades it nucleotide by nucleotide without the exonuclease-poly(A) complex dissociating, whereas the 74 kDa protein operates distributively, with the complex dissociating after one operational step and having to be regenerated.
The poly(A)-specific 3′-exonuclease activity according to the invention can preferably be isolated from animal or human thymus cells, in particular from calf thymus. In general, a whole cell extract is prepared for this purpose, with protein preferably being precipitated from this extract with ammonium sulfate. In this connection, preference is given to the saturation concentration being approx. 45% ammonium sulfate. It has been found to be particularly advantageous in this connection if, before the true protein precipitation, foreign proteins are separated off by being precipitated at a saturation concentration of preferably approx. 25% ammonium sulfate, such that the desired 3′-exonuclease activity can, in a subsequent step, be precipitated out of the supernatant at an ammonium sulfate saturation of approx. 45%. This protein fractionation itself separates off a considerable portion of unwanted foreign proteins.
The precipitate is then subjected to chromatography on a basic anion exchanger, preferably on a weakly basic anion exchanger, in particular on DEAE, such as DEAE-Sepharose. In general, the active fraction elutes at an approx. 0.17 M concentration of a salt, preferably a monovalent salt such as KCl. The eluted active fraction, which has been dialyzed in a customary manner, is then subjected to an affinity chromatography, preferably on heparin, since it has been found, surprisingly, that the active fraction binds particularly well to heparin, e.g. Heparin Sepharose

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