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Analysis of alternative cleavage and polyadenylation by 3' region extraction and deep sequencing

Mainul Hoque1, Zhe Ji, Dinghai Zheng

  • 1Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School, Newark, New Jersey, USA.

Nature Methods
|December 18, 2012
PubMed

Insights

Alternative cleavage and polyadenylation (APA) generates diverse mRNA. A new method, 3'READS, accurately mapped thousands of new poly(A) sites, revealing distinct APA patterns in development and differentiation.

Area of Science:

  • Genomics
  • Molecular Biology
  • RNA Biology

Background:

  • Alternative cleavage and polyadenylation (APA) is a key mechanism for generating mRNA diversity.
  • Accurate identification of poly(A) sites (pAs) is crucial for understanding gene regulation.
  • Existing methods for pA identification are often plagued by mispriming issues.

Purpose of the Study:

  • To develop and validate a novel method for accurate poly(A) site mapping.
  • To comprehensively map poly(A) sites in the mouse genome.
  • To investigate the role of APA in gene regulation during development and differentiation.

Main Methods:

  • Development of 3' region extraction and deep sequencing (3'READS) to overcome mispriming.
  • Application of 3'READS for comprehensive poly(A) site identification in the mouse genome.
  • Quantitative analysis of APA isoform abundance during embryonic development and cell differentiation.

Main Results:

  • Discovery of over 5,000 previously unannotated poly(A) sites, highlighting the need for accurate mapping tools.
  • Identification of distinct APA usage patterns between mRNA and long noncoding RNA genes.
  • Demonstration that promoter-distal poly(A) sites increase in abundance during development and differentiation, with stronger sites correlating with upregulation.

Conclusions:

  • The 3'READS method provides a robust approach for accurate poly(A) site identification.
  • Alternative cleavage and polyadenylation plays a significant role in the regulation of both mRNA and lncRNA genes.
  • Global modulation of 3' end processing activity underlies developmental and differentiation-associated changes in APA isoform usage.

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