Identification of the N-terminal Glycine-arginine Rich (GAR) Domain in Giardia lamblia Fibrillarin and Evidence of its Essentiality for snoRNA Binding
Sumallya Karmakar, Dibyendu Raj, Sandipan Ganguly
International Journal of TROPICAL DISEASE & Health · pp. 318–327 · Published 3 Sep 2013
10.9734/IJTDH/2013/4391Abstract
Aims: Study the role of glycine-arginine rich (GAR) domain of fibrillarin in Giardia lamblia. Study Design: Identifying a specific glycine arginine rich (GAR) sequence of Giardia fibrillarin which plays an important role in ribonucleoprotein particles complex formation with snoRNA during post transcriptional modifications of rRNA. The present study uses Electrophoretic Mobiliy Shift Assay (EMSA) to detect protein-RNA interactions. 32P labeled snoRNA incubated with purified fibrillarin or GAR domain truncated fibrillarin protein were separated by a non denaturing polyacrylamide gel where the band patterns suggested the interaction of the RNAs with both proteins. Place and Duration of Study: Department of Parasitology, National Institute of Cholera and Enteric Diseases, Indian Council of Medical Research, Kolkata, between January 2011 and July 2012. Methodology: Homology analysis of G. lamblia fibrillarin was performed with fibrillarins from Homo sapiens, Mus musculus and Arabidopsis thaliana to determine the conserved domain by ClustalW analysis. Cloning, expression and purification of fibrillarin and GAR domain truncated fibrillarin were done to study the role of GAR domain in snoRNA-fibrillarin binding by EMSA and Gradient EMSA. Immunoblot assay of purified GAR domain truncated fibrillarin was performed by using polyclonal antibody of purified recombinant giardia fibrillarin protein. Results: Gel retardation assay showed that snoRNA does not bind with GAR domain truncated fibrillarin to form any RNP complex. Similarly in gradient EMSA the GAR domain truncated fibrillarin does not bind with any of the in vitro transcribed snoRNA even at much higher concentration than they do for full length purified recombinant fibrillarin. Conclusion: The amino terminal conserved glycine arginine rich (GAR) domain is present in Giardia lamblia fibrillarin and is essential for binding with snoRNA of this protein.
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