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Cytological Approaches to Visualize Intracellular Dynamics of RNA-Binding Proteins at Active Genes in Drosophila

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Poly(ADP-Ribose) Polymerase

Abstract

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including transcription, splicing, localization, translation, and decay of RNA. Interaction of hnRNPs with RNA is a highly dynamic but regulated process. Poly(ADP-ribose) polymerase (PARP)-dependent PARylation of different hnRNPs is a well-known posttranslational modification that affects their interactions with RNA. Here, we described a protocol for in situ localization of RNA-binding proteins (RBPs) on giant polytene chromosomes in Drosophila larval salivary glands, which have been widely used to visualize the dynamic binding profiles of various RBPs and other transcription-related proteins at specific loci on chromosomes. This chapter also includes a stepwise description of RNA:RNA in situ hybridization, in conjunction with immunostaining, using polytene chromosome squashes or intact tissues. We also highlight advanced live cell imaging methods, including FRAP and FLIP, using transgenic lines that express fluorescent-tagged hnRNPs. These cytological approaches can be used to visualize the localization of RNA-binding proteins and their interacting RNAs under different cellular conditions.

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Acknowledgments

We thank Dr. H. Saumweber (Germany) for kind gift of P11 antibody. AKS thanks the Department of Biotechnology, Govt. of India (New Delhi) for the Ramalingaswami Re-entry Fellowship (BT/RHD/35/02/2006). SCL thanks the Science & Engineering Research Board (Govt. of India) for Distinguished Fellowship. We thank the Department of Science & Technology, Govt. of India (New Delhi), and the Banaras Hindu University for Confocal Microscopy facility.

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Correspondence to Anand K. Singh or Subhash C. Lakhotia .

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Singh, A.K., Lakhotia, S.C. (2023). Cytological Approaches to Visualize Intracellular Dynamics of RNA-Binding Proteins at Active Genes in Drosophila. In: Tulin, A.V. (eds) Poly(ADP-Ribose) Polymerase. Methods in Molecular Biology, vol 2609. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2891-1_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2891-1_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2890-4

  • Online ISBN: 978-1-0716-2891-1

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