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The PARN deadenylase targets a discrete set of mRNAs for decay and regulates cell motility in mouse myoblasts

ABSTRACT: Almost all cellular mRNAs terminate in a 3’ poly(A) tail, the removal of which can induce both translational silencing and mRNA decay. Mammalian cells encode many poly(A)-specific exoribonucleases but their individual roles are poorly understood. Here, we undertook an analysis of the role of PARN deadenylase in mouse myoblasts using global measurements of mRNA decay rates. Our results reveal that a discrete set of mRNAs exhibit altered mRNA decay as a result of PARN depletion and that stabilization is associated with increased poly(A) tail length and translation. We determined that stabilization of mRNAs does not generally result in their increased abundance supporting the idea that mRNA decay is coupled to transcription. Importantly, PARN knockdown has wide ranging effects on gene expression that specifically impact the extracellular matrix and cell migration. Finally, although PARN has its own unique target transcripts it also influences some genes whose expression is modulated by other deadenylases.

ORGANISM(S): Mus musculus

PROVIDER: GSE35944 | GEO | 2012/08/31

SECONDARY ACCESSION(S): PRJNA151973

REPOSITORIES: GEO

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Project description:We sought to determine the effect of TbPARN-1 overexpression on global mRNA steady-state levels in T. brucei. The mRNA expression profile of tet-induced TbPARN-1 OvEx procyclic cells was compared to the mRNA profile of control cells (expressing tet-induced TAP tag alone) by microarray analysis. Since overexpression of TbPARN-1 showed increased deadenylation activity in cytoplasmic extracts, overexpressing PARN-1 in vivo should result in more rapid deadenylation and decay of mRNAs targeted by PARN-1. Thus, mRNAs with lower steady state levels in PARN-1 OvEx cells can be considered likely targets for PARN-1-dependent deadenylation. Three different clones of the PARN-1 OvEx and Control cell lines were used for analysis. Each experiment was run in duplicate, with the dye-labeling reversed between duplicates. The T. brucei version 3 microarray chip (PFGRC) was used for this experiment. Each gene on the microarray was represented in duplicate, so for each experiment, mRNA levels were obtained from both points and averaged. As a control between arrays, we used a control probe set obtained from PFGRC. "The probe set consists of Cy3 and Cy5 end labeled 40-mer probes that are complementary to a set of 500 Arabidopsis thaliana 70-mer targets on PFGRC DNA microarrays. The UMSS is supplied as a ready to use mix of the Cy3 and Cy5 labeled oligonucleotides. A small aliquot is spiked into Cy-dye labeled nucleic acid samples just prior to hybridization on PFGRC microarrays. The UMSS produces a set of reference fluorescent signal intensities and ratios that are generated independent of the user-specific experimental samples. Since PFGRC intentionally targeted a heavy representation of Cy3 and Cy5 signals in the mid-range of detection, the Cy3 and Cy5 signal intensities and ratios generated by the UMSS should be highly reproducible. These signals serve as a reference point to judge the success of microarray experiments, and troubleshoot potential problems."

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The PARN deadenylase targets a discrete set of mRNAs for decay and regulates cell motility in mouse myoblasts

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