Fibrillarin Protein, Cat# Prot-r-Fib

Fibrillarin Protein, Cat# Prot-r-Fib

Nop1p was originally identified as a nucleolar protein of bakers yeast, Saccharomyces cerevisiae (accession P15646). The Nop1p protein is 327 amino acids in size (34.5kDa), is essential for yeast viability, and is localized in the nucleoli (1). The systematic name for S. cerevisiae Nop1 is YDL014W, and it is now known to be part of the small subunit processome complex, involved in the processing of pre-18S ribosomal RNA.

Nop1p is the yeast homologue of a protein apparently found in all eukaryotes and archea generally called fibrillarin. Fibrillarin/Nop1p is extraordinarily conserved, so that the yeast and human proteins are 67% identical, and the human protein can functionally replace the yeast protein. This means that suitably cross-reactive antibodies to Nop1p/fibrillarin, like MCA-38F3, can be used to reveal nucleoli and study fibrillarin/Nop1p in all eukaryotes and archea tested to date.

Human fibrillarin has been characterized (accession P22087) and the human fibrillarin gene is located on chromosome 19 (19q13.1). Fibrillarin/Nop1p proteins have been cloned and sequenced from several other species (e.g. Mouse, accession P35550, Xenopus accession P22232, C. elegans accession Q22053, and S. pombe accession P35551. The N terminal ~80 amino acids contain multiple copies based on the peptide RGG, or arginine-glycine-glycine, sometimes referred to as GAR repeats, characteristic of the GAR family of molecules. The remaining ~240 amino acids consist of the so called fibrillarin domain.

A fibrillarin homologue has also been identified in the genome of the archean Methanococcus (accession NC_000909). This protein lacks the RGG rich N-terminal extension but is clearly homologous to the other sequences throughout all of the fibrillarin domain. The 3D structure of this molecule has been determined and shown to consist of 2 extended β-sheets flanked by α-helixes (Medline link). Patients with the autoimmune disease scleroderma often have strong circulating autoantibodies to a ~34 kDa protein which was subsequently found to be fibrillarin. Recent studies show that knock out of the fibrillarin gene in mice results in embryonic lethality, although mice with only one functional fibrillarin/Nop1p gene were viable (3).


Fibrillarin Protein
His-tagged fibrillarin was run out on an SDS-PAGE gel at 1 µg, 0.5 µg in the indicated lanes. BSA was also run at 2 µg, 1 µg and 0.5 µg as indicated. Left most lane contains Biorad SDS-PAGE protein standards of indicated size. The vector adds an N-terminal His-tag which was used to purify the protein and this, along with some other vector derived sequence, adds about 5 kDa to the molecule. This vector derived sequence can be removed if required.

We generated a cDNA encoding the human fibrillarin and expressed this in E. coli. The vector adds an N-terminal His-tag which was use to purify the protein and this, along with some other vector derived sequence, adds about 5 kDa to the molecule. The construct therefore has a total size of about 37 kDa as shown.

References:

1. Ochs RL, Lischwe MA, Spohn WH, Busch H. Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol Cell 54:123-133 (1985).

2. Aris JP and Blobel G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol. 107:17-31 (1988).

3. Newton K, Petfalski E, Tollervey D, Caceres JF. Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse. Mol Cell Biol. 23:8519-8527 (2003).

4. Tyagi S and Alsmadi O. Imaging native beta-actin mRNA in motile fibroblasts. Biophys J. 87:4153-62 (2004).

5. Paeschke1 K, Simonsson T, Postberg J, Rhodes D, Lipps H-J. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo Nature Structural & Molecular Biology 12, 847-854 (2005)

6. Vermaak D, Henikoff S, Malik HS. Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila. PLoS Genetics 1:96-108 (2005).