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Chain termination and inhibition of mammalian poly(A) polymerase by modified ATP analogues.

ABSTRACT: We report the inhibition of mammalian polyadenylation by the triphosphate derivatives of adenosine analogues, 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-amino-Ado), which are under preclinical and clinical investigations for the treatment of hematological malignancies. The nucleotide substrate specificity of bovine poly(A) polymerase (PAP) towards C8-modified ATP analogues was examined using primer extension assays. Radiolabeled RNA primers were incubated with bovine PAP, and in the absence of ATP, no primer extension was observed with 8-Cl-ATP, whereas 8-amino-ATP resulted in chain termination. The effects of modified ATP analogues on ATP-dependent poly(A)-tail synthesis by bovine PAP also were determined, and incubation with analogue triphosphate resulted in significant reduction of poly(A)-tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was evaluated, and polyadenylation of the primer by bovine PAP with ATP was blocked completely. To explain these experimental observations and probe the possible structural mechanisms, molecular modeling was employed to examine the interactions between PAP and various ATP analogues. Molecular docking demonstrated that C8-modifications of ATP led to increased distance between the 3'-hydroxyl group of the RNA oligonucleotide terminus and the alpha-phosphate of ATP that render the molecules in an unfavorable position for incorporation into RNA. Similarly, C8-substitution with a chlorine or amino group at the 3'-terminal residue of RNA also inhibits further chain elongation by PAP. In conclusion, modified ATP analogues may exert their biological effects through polyadenylation inhibition, and thus may provide an RNA-directed mechanism of action for 8-Cl-Ado and 8-amino-Ado.

SUBMITTER: Chen LS

PROVIDER: S-EPMC2812641 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Chain termination and inhibition of mammalian poly(A) polymerase by modified ATP analogues.

Chen Lisa S LS Du-Cuny Lei L Vethantham Vasupradha V Hawke David H DH Manley James L JL Zhang Shuxing S Gandhi Varsha V

Biochemical pharmacology 20091006 5

We report the inhibition of mammalian polyadenylation by the triphosphate derivatives of adenosine analogues, 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-amino-Ado), which are under preclinical and clinical investigations for the treatment of hematological malignancies. The nucleotide substrate specificity of bovine poly(A) polymerase (PAP) towards C8-modified ATP analogues was examined using primer extension assays. Radiolabeled RNA primers were incubated with bovine PAP, and in the ab ...[more]

PMID: 19814999

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Project description:Making the correct choice between transcription elongation and transcription termination is essential to the function of RNA polymerase II, and fundamental to gene expression. This choice can be influenced by factors modifying the transcription complex, factors modifying chromatin, or signals mediated by the template or transcript. To aid in the study of transcription elongation and termination we have developed a transcription elongation reporter system that consists of tandem luciferase reporters flanking a test sequence of interest. The ratio of expression from the reporters provides a measure of the relative rates of successful elongation through the intervening sequence.Size matched fragments containing the polyadenylation signal of the human beta-actin gene (ACTB) and the human beta-globin gene (HBB) were evaluated for transcription termination using this new ratiometric tandem reporter assay. Constructs bearing just 200 base pairs on either side of the consensus poly(A) addition site terminated 98% and 86% of transcription for ACTB and HBB sequences, respectively. The nearly 10-fold difference in read-through transcription between the two short poly(A) regions was eclipsed when additional downstream poly(A) sequence was included for each gene. Both poly(A) regions proved very effective at termination when 1100 base pairs were included, stopping 99.6% of transcription. To determine if part of the increased termination was simply due to the increased template length, we inserted several kilobases of heterologous coding sequence downstream of each poly(A) region test fragment. Unexpectedly, the additional length reduced the effectiveness of termination of HBB sequences 2-fold and of ACTB sequences 3- to 5-fold.The tandem construct provides a sensitive measure of transcription termination in human cells. Decreased Xrn2 or Senataxin levels produced only a modest release from termination. Our data support overlap in allosteric and torpedo mechanisms of transcription termination and suggest that efficient termination is ensured by redundancy.

Dataset Information

Chain termination and inhibition of mammalian poly(A) polymerase by modified ATP analogues.

Publications

Chain termination and inhibition of mammalian poly(A) polymerase by modified ATP analogues.

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