Two RNA-binding sites in plant fibrillarin provide interactions with various RNA substrates

Published online 23 July 2011 Nucleic Acids Research, 2011, Vol. 39, No. 20 8869–8880 doi:10.1093/nar/gkr594 Two RNA-binding sites in plant fibrillarin provide interactions with various RNA substrates D. V. Rakitina1, Michael Taliansky2,*, J. W. S. Brown2,3 and N. O. Kalinina1 1 Department of Virology and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia, 2The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK and 3 Plant Sciences Division, University of Dundee, DD2 5DA, UK Received December 28, 2010; Revised July 1, 2011; Accepted July 4, 2011 ABSTRACT Fibrillarin, one of the major proteins of the nucleolus, plays several essential roles in ribosome biogenesis including pre-rRNA processing and 20 -O-ribose methylation of rRNA and snRNAs. Recently, it has been shown that fibrillarin plays a role in virus infections and is associated with viral RNPs. Here, we demonstrate the ability of recombinant fibrillarin 2 from Arabidopsis thaliana (AtFib2) to interact with RNAs of different lengths and types including rRNA, snoRNA, snRNA, siRNA and viral RNAs in vitro. Our data also indicate that AtFib2 possesses two RNA-binding sites in the central (138–179 amino acids) and C-terminal (225–281 amino acids) parts of the protein, respectively. The conserved GCVYAVEF octamer does not bind RNA directly as suggested earlier, but may assist with the proper folding of the central RNA-binding site. INTRODUCTION Fibrillarin is a 33–36 kDa nucleolar protein involved in multiple aspects of RNA biogenesis. It is a major protein in the fibrillar regions of the nucleolus where rRNA transcription and early pre-rRNA processing take place (1,2). Fibrillarin is also found in Cajal bodies (CBs), subnuclear dynamic particles that contain distinct components involved in RNA transcription and editing such as mRNA splicing and small nuclear/nucleolar ribonucleoprotein (sn/snoRNP) biogenesis (3,4). Fibrillarin is a core component of box C/D snoRNP particles, which are responsible for site-specific 20 -O-ribose methylation of rRNA and for pre-rRNA cleavage, and box C/D-containing small CB-specific RNPs (scaRNPs) that methylate snRNAs (5,6). Assembled sn/snoRNPs traffic through CBs before accumulating in both splicing speckles (sites of storage/assembly of splicing factors/complexes) and also in the dense fibrillar component of the nucleolus where early pre-rRNA processing events take place. In all cases, fibrillarin is found in multi-component complexes containing RNA (snoRNA, pre-rRNA) and proteins. In eukaryotes, the mature box C/D snoRNP complex competent for snRNA/pre-rRNA modification contains the RNA-binding 15.5-kDa protein and the two related proteins Nop56 and Nop58 in addition to fibrillarin and the snoRNA (5). Fibrillarin is highly evolutionarily conserved with respect to sequence, structure and function (5,7–10). On the basis of the amino acid sequence alignment of fibrillarin proteins from different species, Aris and Blobel (7) suggested that fibrillarin contains several domains. Within the human fibrillarin protein (36 kDa—320 amino acids), three structure/functions regions have been predicted (7; Figure 1). The N-terminal region of fibrillarin is represented by a glycine- and arginine-rich (GAR) domain that is also found in fibrillarin from Saccharomyces cerevisiae, Xenopus laevis and Tetrahymena thermophila (7,9,11), but not in archaeal fibrillarin proteins (5,8). The GAR domain is methylated at arginine residues (12), possibly by the arginine methyltransferases found in complex with fibrillarin (13). The GAR domain is responsible for the interactions with various cellular (14,15) and viral proteins (16,17). The data concerning the role of the GAR domain in nucleolar localization are contradictory. Some findings demonstrate that the GAR domain is not required to target fibrillarin to the nucleolus or CBs, although it contains a nucleolar localization signal (3), but other results suggest that the GAR domain provides the nucleolar targeting of fibrillarin in plants and human cells (9,18). Since the central part of the protein contains 90 amino acid residues with some similarity to the RNA recognition motif (RRM) present in various RNA-binding proteins, Aris and Blobel (7) have named this region a ‘putative RNA-binding domain’. The alignment with several RNA-binding proteins allowed them to identify an *To whom correspondence should be addressed. Tel: +44(0)1382562731; Fax: +44 (0)1382 562426; Email: ß The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 8870 Nucleic Acids Research, 2011, Vol. 39, No. 20 A GAR-region 1 spacer 77 "R region" 138 "α-rich region" [GCVYAVEF] [180-187] 225 320 a/a AtFib2 N-terminal domain MTase domain B AtFib2ΔGAR AtFib2ΔR AtFib2Δα−rich AtFib2GAR AtFib2GAR-spacer AtFib2-Ala 8xAla AtFib2R 8xAla AtFib2R-Ala AtFib2R138-179 AtFib2R188-224 AtFib2R138-187 AtFib2α-rich AtFib2α-rich225-281 AtFib2α-rich282-320 Figure 1. Schematic representation of AtFib2 protein (A) and its deletion mutants (B). The main fibrillarin regions predicted by Aris and Blobel (7) for human fibrillarin are as follows: N-terminal glycine–arginine-rich region (GAR), central region (referred to as R-region) that was suggested to provide RNA-binding activity via the conserved amino acid octamer GLVYAVEF, the spacer between these regions (spacer) and the C-terminal region containing predicted a-helices (referred to as a-rich-region). Domains identified by Wang et al. (8) for fibrillarin protein of Methanocaldococcus jannaschii are the N-terminal domain (mostly overlaps with GAR region) and the MTase domain comprising the central R-region and C-terminal a-rich region. The conserved GCVYAVEF octamer is indicated with vertical lines, and when replaced with eight alanines sequence (8 Ala)—with white dots on black. The positions of AtFib2 deletion mutants are indicated. Internal deletion in AtFib2
R mutant is shown by a dashed line. octameric region, which resembles a classical RNP1 motif within the RRM that directly participates in RNA-binding, (R/K)-G-(F/Y)-(G/A)-(F/Y)-(I/L/V)-X(F/Y) (19). This is conserved among human, S. cerevisae and X. laevis fibrillarin proteins as a GLVYAVEF sequence (7). The C-terminal region of the protein contains a short sequence (33 amino acid residues) predicted to form an a-helix structure. The C-terminal region of fibrillarin targets it to CBs (3,18) and interacts directly with Nop56 (20). Together, the central and C-terminal regions of the protein constitute a conserved Ado-Met-dependent methyltransferase (MTase)-like domain that contains S-adenosyl methionine (SAM, the methionine group donor) binding moti (...truncated)