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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Krecic AM, Swanson MS (1999) hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol 11(3):363–371
Piccolo LL, Corona D, Onorati MC (2014) Emerging roles for hnRNPs in post-transcriptional regulation: what can we learn from flies? Chromosoma 123(6):515–527. https://doi.org/10.1007/s00412-014-0470-0
Dreyfuss G, Kim VN, Kataoka N (2002) Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol 3(3):195–205. https://doi.org/10.1038/nrm760
Singh AK, Lakhotia SC (2016) The hnRNP A1 homolog Hrb87F/Hrp36 is important for telomere maintenance in Drosophila melanogaster. Chromosoma 125(3):373–388. https://doi.org/10.1007/s00412-015-0540-y
Geuens T, Bouhy D, Timmerman V (2016) The hnRNP family: insights into their role in health and disease. Hum Genet 135(8):851–867. https://doi.org/10.1007/s00439-016-1683-5
Ji Y, Tulin AV (2009) Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins modulates splicing. Nucleic Acids Res 37(11):3501–3513. https://doi.org/10.1093/nar/gkp218
Pinnola A, Naumova N, Shah M, Tulin AV (2007) Nucleosomal core histones mediate dynamic regulation of poly(ADP-ribose) polymerase 1 protein binding to chromatin and induction of its enzymatic activity. J Biol Chem 282(44):32511–32519. https://doi.org/10.1074/jbc.M705989200
Roy JK, Lakhotia SC (2021) Study of polytene chromosomes of Drosophila melanogaster. In: Lakhotia SC, Ranganath HA (eds) Experiments with Drosophila for biology courses. Indian Academy of Sciences, Bengaluru, pp 175–183
Chaudhary GR, Pandey A, Singh A, Yadav V, Dwivedi V, Arya R, Lakhotia SC (2021) Rearing and handling of Drosophila – a primer for laboratory experiments. In: Lakhotia SC, Ranganath HA (eds) Experiments with Drosophila for biology courses. Indian Academy of Sciences, Bengaluru, pp 1–31
Saumweber H, Symmons P, Kabisch R, Will H, Bonhoeffer F (1980) Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster: establishment of antibody producing cell lines and partial characterization of corresponding antigens. Chromosoma 80(3):253–275
Yao J, Zobeck KL, Lis JT, Webb WW (2008) Imaging transcription dynamics at endogenous genes in living Drosophila tissues. Methods 45(3):233–241. https://doi.org/10.1016/j.ymeth.2008.06.004
Lakhotia SC, Tapadia MG (1998) Genetic mapping of the amide response element(s) of the hsr omega locus of Drosophila melanogaster. Chromosoma 107(2):127–135. https://doi.org/10.1007/s004120050288
Prasanth KV, Rajendra TK, Lal AK, Lakhotia SC (2000) Omega speckles – a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila. J Cell Sci 113(Pt 19):3485–3497
Singh AK, Lakhotia SC (2015) Dynamics of hnRNPs and omega speckles in normal and heat shocked live cell nuclei of Drosophila melanogaster. Chromosoma 124(3):367–383. https://doi.org/10.1007/s00412-015-0506-0
Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16(9):1055–1069. https://doi.org/10.1016/S0006-3495(76)85755-4
Klonis N, Rug M, Harper I, Wickham M, Cowman A, Tilley L (2002) Fluorescence photobleaching analysis for the study of cellular dynamics. Eur Biophys J 31(1):36–51. https://doi.org/10.1007/s00249-001-0202-2
Phair RD, Misteli T (2000) High mobility of proteins in the mammalian cell nucleus. Nature 404(6778):604–609. https://doi.org/10.1038/35007077
Singh AK, Choudhury SR, De S, Zhang J, Kissane S, Dwivedi V, Ramanathan P, Petric M, Orsini L, Hebenstreit D, Brogna S (2019) The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from gene loci. elife 8. https://doi.org/10.7554/eLife.41444
Nissim-Rafinia M, Meshorer E (2011) Photobleaching assays (FRAP & FLIP) to measure chromatin protein dynamics in living embryonic stem cells. J Vis Exp 52. https://doi.org/10.3791/2696
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.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2891-1_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2890-4
Online ISBN: 978-1-0716-2891-1
eBook Packages: Springer Protocols