Depletion of Nuclear Poly(A) Binding Protein PABPN1 Produces a Compensatory Response by Cytoplasmic PABP4 and PABP5 in Cultured Human Cells

Abstract

BackgroundIn vertebrates, poly(A) binding protein (PABP) is known to exist in five different isoforms. PABPs are primarily cytosolic with the exception of the nuclear PABP (PABPN1), which is located in the nucleus. Within the nucleus, PABPN1 is believed to bind to the poly(A) tail of nascent mRNA and along with cleavage and polyadenylation specificity factor (CPSF) define the length of the newly synthesized poly(A) tail.Methodology/Principal FindingsThe cellular role of PABP1 has been extensively studied over the years; however, the function of other PABPs remains poorly defined. In order to understand the role of PABPN1 in cellular mRNA metabolism and it’s interrelation with other PABPs, we depleted PABPN1 using RNAi in HeLa and HEK293 cells. Our results show that PABPN1 depletion did not have any effect on the poly(A) tail length, nuclear export of mRNA, mRNA translation, and transcription. Rather, PABPN1 depletion resulted in a compensatory response as observed by increased level of PABP5 and nuclear accumulation of PABP4. In addition, PABP4 was associated with the poly(A) tract of pre-mRNA and CPSF in PABPN1 depleted cells. Nevertheless, PABPN1 depletion significantly affected cell survival as evidenced by an increase in apoptosis markers: phosphorylated p53 and PUMA and as judged by the expression of ER stress marker GRP78.ConclusionOur results suggest that although function of PABPN1 may be compensated by nuclear translocation of PABP4 and perhaps by increase in the cytoplasmic abundance of PABP5, these were not sufficient to prevent apoptosis of cells. Thus PABPN1 may have a novel anti apoptotic role in mammalian cells.

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

“…This model is supported by several in vivo observations, including a modest decrease in steady-state poly(A) tail length upon knockdown of PABPN1 in primary mouse myoblasts (Chen et al , 1999b ;Benoit et al , 2005 ;Apponi et al , 2010 ). However, because the knockdown of PABPN1 had no effect on steady-state poly(A) tail length in two studies using other cells (Beaulieu et al , 2012 ;Bhattacharjee and Bag , 2012 ), a role of the protein in general mRNA polyadenylation has recently been challenged (Beaulieu et al , 2012 ). Possible explanations for the lack of an effect include compensatory roles of other poly(A)-binding proteins (Bhattacharjee and Bag , 2012 ) and, as with any RNAi experiment, insufficient depletion.…”

“…PABPN1, together with the heterooligomeric cleavage and polyadenylation specificity factor (CPSF), increases the affinity of poly(A) polymerase for the RNA to be elongated, apparently by a direct contact with the enzyme. Increased RNA affinity of poly(A) polymerase results in increased processivity of polyadenylation and, consequently, a dramatically enhanced rate of elongation (Wahle , 1991 ;Bienroth et al , 1993 ;Kerwitz et al , 2003 ). After the polymerization of 200 -250 nucleotides, processive elongation is terminated because the interaction between CPSF and poly(A) polymerase can no longer be maintained.…”

“…However, because the knockdown of PABPN1 had no effect on steady-state poly(A) tail length in two studies using other cells (Beaulieu et al , 2012 ;Bhattacharjee and Bag , 2012 ), a role of the protein in general mRNA polyadenylation has recently been challenged (Beaulieu et al , 2012 ). Possible explanations for the lack of an effect include compensatory roles of other poly(A)-binding proteins (Bhattacharjee and Bag , 2012 ) and, as with any RNAi experiment, insufficient depletion. Most importantly, the in vitro data demonstrate an effect of PABPN1 on the rate of poly(A) tail synthesis, which has not been examined in vivo under conditions of PABPN1 depletion; whether or not there should be an effect on the final length, and thus steady-state length distribution, cannot be predicted with certainty.…”

Shutdown of Achaete-scute Homolog-1 Expression by Heterogeneous Nuclear Ribonucleoprotein (hnRNP)-A2/B1 in Hypoxia

“…5, e and f). Although this suggests that PABP2 does not play a role in regulating hASH1 levels under these conditions, the lack of effect might also be explained by the fact that PABP4 and PABP5 can compensate for the function of PABP2 (41).…”

Poly(A) Tail-Mediated Gene Regulation by Opposing Roles of Nab2 and Pab2 Nuclear Poly(A)-Binding Proteins in Pre-mRNA Decay

“…Similarly, Nab2 does not appear to be required to stimulate the elongation process of poly(A) tail synthesis in budding yeast (27). Recent data also indicate that the large majority of human protein-coding genes express normal levels of polyadenylated mRNA in PABPN1-deficient cells (11,54). These observations question the need for nuclear PABPs to load onto nascent poly(A) tails to stimulate PAP activity during mRNA polyadenylation in vivo.…”