Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

Zannat, Mst. Thangima; Bhattacharjee, Rumpa Biswas; Bag, Jnanankur

doi:10.1016/j.bbrc.2011.04.003

Cited by 6 publications

(6 citation statements)

References 15 publications

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“…A gain-of-function of ATXN2 might also underlie the reduced weight of the knock-in animals, since ATXN2 knock-out mice are known to have excessive weight [47]. Specifically in cerebellar tissue, the insolubility and sequestration led to a deficiency in soluble PABPC1 levels, an effect that might result in impaired RNA processing as well as protein synthesis [50] and therefore possibly critical for the Purkinje neurons with their large ribosomal machinery. Indeed, these molecular abnormalities correlate temporally with the late-onset appearance of motor deficit.…”

Section: Discussionmentioning

confidence: 99%

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ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

et al. 2012

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Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8.

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Section: Discussionmentioning

confidence: 99%

“…The depletion of PABPC1 was shown to prevent global protein synthesis and to promote cell death in mammalian cells [50]. PABPC1 binds to the 3′ poly(A) tail of mRNAs to mediate their circularization which precedes ribosome recruitment and translation initiation [51].…”

Section: Discussionmentioning

confidence: 99%

ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

et al. 2012

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“…Western blotting, immunostaining, and in situ hybridization were performed as described previously [22], [23]. After immunostaining with PABP4 antibody, the nuclei of the cells were further stained with DAPI (Santa Cruz Biotechnology, CA, USA) (0.5 µg/µl) for 10 min, washed twice with PBS and mounted onto slides.…”

Section: Methodsmentioning

confidence: 99%

“…An aliquot of total RNA (100–500 ng) was reverse transcribed using High Capacity cDNA transcription kit (Applied Biosystems, Life Technologies). After the reaction, 2 µL of the cDNA sample was amplified by PCR in a total master mix (Fermentas, Amherst, NY, USA) reaction volume of 50 µL, which included 100 ng of primers specific for diffeent mRNAs (Sigma, Oakville, ON, Canada) [22]. Primers used have been described previously [22], [24].…”

Section: Methodsmentioning

confidence: 99%

“…After the reaction, 2 µL of the cDNA sample was amplified by PCR in a total master mix (Fermentas, Amherst, NY, USA) reaction volume of 50 µL, which included 100 ng of primers specific for diffeent mRNAs (Sigma, Oakville, ON, Canada) [22]. Primers used have been described previously [22], [24]. The amplification was performed using an initial denaturation step at 95°C for 4 min, and depending on the cellular level of the individual mRNA, it was followed by 30–35 cycles of denaturation at 95°C for 20 s, annealing ranging from 55–60°C, depending on the primer for 20 s and extension at 72°C for 20 s. Samples were analyzed by 1% agarose gel electrophoresis and the relative expression values of all mRNAs were normalized by the β-actin mRNA level.…”

Section: Methodsmentioning

confidence: 99%

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

Bhattacharjee

Bag

2012

PLoS ONE

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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.

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Cerebellar ataxia and functional genomics: Identifying the routes to cerebellar neurodegeneration

Smeets

Verbeek

2014

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Cerebellar ataxias are progressive neurodegenerative disorders characterized by atrophy of the cerebellum leading to motor dysfunction, balance problems, and limb and gait ataxia. These include among others, the dominantly inherited spinocerebellar ataxias, recessive cerebellar ataxias such as Friedreich's ataxia, and X-linked cerebellar ataxias. Since all cerebellar ataxias display considerable overlap in their disease phenotypes, common pathological pathways must underlie the selective cerebellar neurodegeneration. Therefore, it is important to identify the molecular mechanisms and routes to neurodegeneration that cause cerebellar ataxia. In this review, we discuss the use of functional genomic approaches including whole-exome sequencing, genome-wide gene expression profiling, miRNA profiling, epigenetic profiling, and genetic modifier screens to reveal the underlying pathogenesis of various cerebellar ataxias. These approaches have resulted in the identification of many disease genes, modifier genes, and biomarkers correlating with specific stages of the disease. This article is part of a Special Issue entitled: From Genome to Function.

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Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

Cited by 6 publications

References 15 publications

ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

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

Cerebellar ataxia and functional genomics: Identifying the routes to cerebellar neurodegeneration

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