Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells

Joana Wilton, Michael Tellier, Takayuki Nojima, Angela M. Costa, Maria Jose Oliveira, Alexandra Moreira

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Transcription termination in eukaryotic cells involves the recognition of polyadenylation signals (PAS) that signal the site of pre-mRNA cleavage and polyadenylation. Most eukaryotic genes contain multiple PAS that are used by alternative polyadenylation (APA), a co-transcriptional process that increases transcriptomic diversity and modulates the fate of the mRNA and protein produced. However, current tools to pinpoint the relationship between mRNAs in different subcellular fractions and the gene expression outcome are lacking, particularly in primary human immune cells, which, due to their nature, are challenging to study. Here, we describe an integrative approach using subcellular fractionation and RNA isolation, chromatin-bound and nucleoplasmic RNA-Sequencing, 3′ RNA-Sequencing and bioinformatics, to identify accurate APA mRNA isoforms and to quantify gene expression in primary human macrophages. Our protocol includes macrophage differentiation and polarization, co-culture with cancer cells, and gene silencing by siRNA. This method allows the simultaneous identification of macrophage APA mRNA isoforms integrated with the characterization of nuclear APA events, the identification of the molecular mechanisms involved, as well as the gene expression alterations caused by the cancer-macrophage crosstalk. With this methodology we identified macrophage APA mRNA signatures driven by the cancer cells that alter the macrophage inflammatory and transcriptomic profiles, with consequences for macrophage physiology and tumor evasion.

Original languageEnglish
Title of host publicationmRNA 3' End Processing and Metabolism
EditorsBin Tian
PublisherAcademic Press Inc.
Pages349-399
Number of pages51
ISBN (Print)9780128235737
DOIs
Publication statusPublished - Jan 2021

Publication series

NameMethods in Enzymology
Volume655
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells'. Together they form a unique fingerprint.

Cite this