Wilhelm bernhard workshop on the cell nucleus

SIGNAL SEQUENCES IN THE MOLECULE OF THE NUCLEOLAR PROTEIN FIBRILLARIN DIRECTING ITS SPECIFIC LOCALIZATION WITHIN THE NUCLEOLUS

^1,3Mukharyamova K.Sh., ²Levitsky S.A., ²Veiko V.P., and ^1,3Zatsepina O.V.

¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAN, ²Institute of Genetics and Selection of Industrial Microorganisms of The Ministry of Economics, and ³A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Moscow, Russia
Fibrillarin (321 amino acids in human) is a key nucleolar protein that is essential for posttranscriptional modifications of rRNA transcripts, including pre-rRNA processing, pre-rRNA methylation and ribosome assembly. Human fibrillarin has three structural domains. NH₂-terminus (~80 amino acids) contains a glycine- and arginine-rich domain (GAR-domain). A central region (~90 amino acids) resembles an RNA-binding (RB) domain that is also present in many small nuclear snRNP proteins. The COOH-terminus contains a small (~30 amino acids) alpha helix domain and apparently has a methyltransferase activity. The domains are separated by the spacer regions (~50 amino acids each), called 1^st and 2^nd. In the present work we studied implication of these domains in specific localization of fibrillarin in the foci of early rRNA synthesis, which can be visualized by run-on transcription assay with BrUTP as precursor. A plasmid encoding full-length fibrillarin/GFP (green fluorescent protein) was kindly donated by Dr. M.O.J. Olson (UMMC, USA) and used for generation of mutant fibrillarin constructs fused to GFP, including pΔ2α/GFP, pGAR1/GFP, pΔRB/GFP, p2α/GFP, pSRB/GFP. HeLa cells were transiently transfected of these plasmids and distribution of fibrillarin/GFP was recorded by fluorescence microscopy after 15-48 h and co-localized with endogenous fibrillarin. Our data showed that a deletion or mutation in any known fibrillarin domain inhibits protein specific (i.e., within the Br-RNA positive foci) localization within the nucleolus. However, together GAR-domain and the 1^st spacer were sufficient to target fibrillarin to the nucleolus. The results are discussed in terms of relevant literature data.

The research was supported by the Russian Foundation of Basic Researches (grant 03-04-48951).