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Yeast Systems Biology

Volume 759 of the series Methods in Molecular Biology pp 133-148

Date:

Polyadenylation State Microarray (PASTA) Analysis

  • Traude H. BeilharzAffiliated withDepartment of Biochemistry and Molecular Biology, Monash University
  • , Thomas PreissAffiliated withGenome Biology Department, The John Curtin School of Medical Research, The Australian National University Email author 

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Abstract

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

Key words

Poly(A) tail polyadenylation deadenylation post-transcriptional regulation translational control mRNA stability polysome microarray transcriptome proteome