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Approaches for Studying mRNA Decay Mediated by SIDER2 Retroposons in Leishmania

  • Barbara PapadopoulouEmail author
  • Michaela Müller-McNicoll
  • Prasad K. Padmanabhan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1201)

Abstract

Regulated mRNA turnover is a highly important process in the control of gene expression in Leishmania and related trypanosomatid protozoa, as these organisms lack control at the level of transcription initiation. A large number of Leishmania transcripts harbor in their 3′UTRs two phylogenetically distinct subfamilies of extinct Short Interspersed DEgenerate Retroposons (SIDER1 and SIDER2) that are involved in posttranscriptional regulation of gene expression. We have shown recently that members of the SIDER2 subfamily promote mRNA destabilization and that degradation of SIDER2-containing mRNAs is initiated by site-specific endonucleolytic cleavage within the second 79-nt SIDER2 signature sequence without prior shortening of the poly(A) tail. Here, we describe experimental procedures for studying the mechanism of SIDER2-mediated mRNA decay. These include RNase protection assays to identify in vivo-generated mRNA decay intermediates following endonucleolytic cleavage, primer extension analysis to precisely map the site(s) of cleavage within SIDER2, and deadenylation assays to assess the polyadenylation state of unstable SIDER2-containing mRNAs in Leishmania.

Key words

Leishmania mRNA decay SIDER2 retroposons Endonucleolytic cleavage RNase protection Primer extension Deadenylation assay 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Barbara Papadopoulou
    • 1
    • 2
    Email author
  • Michaela Müller-McNicoll
    • 3
    • 4
  • Prasad K. Padmanabhan
    • 5
  1. 1.Research Center in Infectious DiseaseCHU de Quebec Research CenterQuebecCanada
  2. 2.Department of Microbiology Infectious Disease and Immunology, Faculty of MedicineLaval UniversityQuebecCanada
  3. 3.Institute for Cell Biology and NeuroscienceGoethe University FrankfurtFrankfurtGermany
  4. 4.Goethe University FrankfurtFrankfurtGermany
  5. 5.Department of Immunology and Infectious DiseasesHarvard School of Public HealthBostonUSA

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