Biological activity and redistribution of nucleolar proteins of two different cell lines treated with cis-dichloro-1,2-propylenediamine-N,N,N',N'-tetraacetato ruthenium (III) (RAP).

The interaction of a newly synthesized antitumor complex cis-dichloro-1,2-propylenediamine-N,N,N',N'-tetraacetato ruthenium (III) (RAP) with DNA was investigated in vitro through a number of techniques including comet assay, immunoprecipitation, and immunolocalization of certain nucleolar proteins (the upstream binding factor (UBF) and fibrillarin) involved in DNA transcription, rRNA processing, and ribosomal assembly. The results showed that RAP binds to the DNA of two cell lines (H4 and Hs-683) causing a delay in cell proliferation rate leading to a number of cellular modifications. These modifications include DNA-damage assessed by the single cell gel electrophoresis method (comet assay) and variation in the expression of nucleolar proteins; UBF was more abundant in RAP treated cells, this was explained by the high affinity of this protein to DNA modified by RAP. On the other hand, fibrillarin was found in less quantities in RAP treated cells which was explained by a de-regulation of the ribosomal machinery caused by RAP.

Identification of the N-terminal Glycine-arginine Rich (GAR) Domain in Giardia lamblia Fibrillarin and Evidence of its Essentiality for snoRNA Binding.

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.