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- Kyriakopoulou, Christina B., et al.
(författare)
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A novel nuclear human poly(A) polymerase (PAP), PAPγ
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:36, s. 33504-33511
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Tidskriftsartikel (refereegranskat)abstract
- Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. Here we show that the 90-kDa isoform is the product of the gene PAPOLG, which is distinct from the previously identified genes for poly(A) polymerases. The 90-kDa isoform is referred to as human PAPγ (hsPAPγ). hsPAPγ shares 60% identity to human PAPII (hsPAPII) at the amino acid level. hsPAPγ exhibits fundamental properties of a bona fidepoly(A) polymerase, specificity for ATP, and cleavage and polyadenylation specificity factor/hexanucleotide-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of hsPAPII. Mutational analysis and sequence comparison revealed that hsPAPγ and hsPAPII have similar organization of structural and functional domains. hsPAPγ contains a U1A protein-interacting region in its C terminus, and PAPγ activity can be inhibited, as hsPAPII, by the U1A protein. hsPAPγ is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on this and previous studies, it is obvious that multiple isoforms of PAP are generated by three distinct mechanisms: gene duplication, alternative RNA processing, and post-translational modification. The exclusive nuclear localization of hsPAPγ establishes that multiple forms of PAP are unevenly distributed in the cell, implying specialized roles for the various isoforms.
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| 4. |
- Nordvarg, Helena, 1969-
(författare)
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Functional Significance of Multiple Poly(A) Polymerases (PAPs)
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 3’ end cleavage and polyadenylation are important steps in the maturation of eukaryotic mRNAs. Poly(A) polymerase (PAP), the enzyme catalysing the addition of adenosine residues, exists in multiple isoforms. In this study the functional significance of multiple poly(A) polymerases have been investigated. It is concluded (i) that at least three mechanisms generate the multiple isoforms i.e. gene duplication, post-translational modification and alternative mRNA processing and (ii) that the different isoforms of poly(A) polymerases have different catalytic properties. The study highlights regulation of poly(A) polymerase activity through modulation of its affinity for the substrate as visualised by the KM parameter. We suggest that trans-acting factors modulating the KM of poly(A) polymerase will play important roles in regulating its activity.A new human poly(A) polymerase (PAPγ) encoded by the PAPOLG gene was identified. PAPγ is 65% homologous to the previously identified PAP. In human cells three isoforms of poly(A) polymerases being 90, 100 and 106 kDa in sizes are present. These native isoforms were purified. The PAPOLA gene encoded the 100 and 106 kDa isoforms while the 90 kDa isoform was encoded by the PAPOLG gene. Native PAPγ was found to be more active than 100 kDa PAP while the hyperphosphorylated 106 kDa PAP isoform was comparably inactive due to a 500-fold decrease in affinity for the RNA substrate. The PAPOLG gene was shown to encode one unique mRNA while the PAPOLA gene generated five different PAP mRNAs by alternative splicing of the last three exons. The PAPOLA encoded mRNAs were divided into two classes based on the composition of the last three exons. Poly(A) polymerases from the two classes were shown to differ in polyadenylation activities. These differences revealed two novel regulatory motifs in the extreme C-terminal end of PAP, one being inactivating and the other activating for polyadenylation activity.
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