Supplemental Data: Analysis of the L7 Gene Family

Supplemental Data: Analysis of the L7 Gene Family

Eukaryotic L7 r-protein is an ortholog of L30 of archaebacteria and eubacteria (Wool et al., 1995). Eubacterial L30 is comprised of approximately 60 amino acid residues, archaebacterial L30 is comprised of approximately 150 amino acid residues, whereas eukaryotic L7 is comprised of approximately 250 amino acid residues (Mizuta et al., 1992). Proteolytic studies and biotinylation experiments have demonstrated that the N-terminal region of L7 is localized on the surface of the 60S subunit whereas the C-terminal region is buried within the ribosome (Lin, 1991; Marion and Marion, 1987). Furthermore, the N-terminus of rat and human L7 contains a motif similar to the consensus sequence of a bZIP (basic-region-leucine-zipper) DNA-binding domain (Hemmerich et al., 1993). In humans, the bZIP region of L7 binds mRNA, inhibits translation of reporter mRNAs, and thus is believed to have a regulatory role in translation (Neumann et al., 1995). There is an Arg and Lys-rich region within the bZIP-like region that is sufficient for RNA binding (Hemmerich et al., 1997). von Mikecz et al. (1999) named the arginine-rich domain within the bZIP region of the N-terminus nucleic acid-binding domain 1 (NBD 1) and demonstrated that human L7 contains an additional nucleic acid-binding domain (NBD 2), which corresponds to the 50 C-terminal amino acids of the protein. NBD 2 does not contain any known nucleic acid binding motifs, but preferentially binds to 28S rRNA, suggesting this region is involved in the attachment of L7 to the large ribosomal subunit (von Mikecz et al., 1999).

A preliminary alignment of Arabidopsis, potato (Solanum tuberosum), rat, Drosophila, and yeast L7 sequences (data not shown) revealed significant sequence conservation among eukaryotes. The alignment shown below (panel A) demonstrates the sequence conservation among the rat and Arabidopsis L7 proteins.The 50 C-terminal amino acid residues that correspond to NBD-2 in human L7 is underlined. The shaded residues indicate residues that are strictly conserved (shaded red) or contain very conservative substitutions (V/I/L; Y/F; K/R; shaded gray) in the alignment of eukaryotic L7 sequences mentioned above.

The evolutionary relatedness of the Arabidopsis L7 proteins was examined by calculating pairwise uncorrected distances.The table illustrates that the Arabidopsis L7 proteins fall into two distinct categories, L7B, C and D (Type I) and L7A (Type II).Within-group distances [L7B to L7C (0.040), L7B to At L7D (0.060), and L7C to L7D (0.027)] are substantially less than between-group distances [L7A to B, C or D, 0.493, 0.500, or 0.513, respectively]. The distance between rat L7 and Arabidopsis L7A (0.560) is distinct from that of rat L7 to Arabidopsis L7B, C, or D (0.267, 0.260 or 0.267, respectively) suggesting that L7A is distinct from L7B, C or D and most likely represents an additional class of L7 proteins.

To further study the evolutionary relatedness of the Arabidopsis L7 proteins with respect to other L7 proteins, maximum parsimony and neighbor joining methods of phylogenetic analyses were utilized. Neighbor joining analysis based on 10 polypeptide sequences, including two plant species (Arabidopsis and potato), rat, Drosophila, yeast and one archaebacterial sequence, resulted in the phylogenetic tree shown below. The L7 polypeptide sequences of plants fall into two categories, one containing Arabidopsis L7B, C, D and potato L7-1 (Type I) and one containing Arabidopsis L7A and potato L7-2 (Type II), further confirming the two types of plant L7 proteins. The classification of the Type I L7 proteins in Arabidopsis and potato is strongly supported (parsimony and uncorrected distance bootstrap % = 97 and 100, respectively). The grouping of rat, Drosophila, and plant Type I L7 proteins is also well supported (parsimony and uncorrected distance bootstrap % = 76 and 96, respectively) suggesting that the duplication event and the subsequent divergence that lead to the Type II L7 proteins (in Arabidopsis and potato) occurred in the plant lineage. Analysis of the GenBank EST database revealed that Type I and II L7 proteins are also maintained in rice, tomato and barley.

References

Hemmerich P, von Mikecz A, Neumann F, Soezeri O, Wolff-Vorbeck G, Zoebelein R, Krawinkel U (1993) Structural and functional properties of ribosomal protein L7 from humans and rodents. Nuc Acids Res 21: 223-231

Hemmerich P, Bosbach S, Von Mikecz A, Krawinkel U (1997) Human ribosomal protein L7 binds RNA with an alpha-helical arginine-rich and lysine-rich domain. Eur J Biochem 245: 549-556

Lin A (1991) Localization of surface peptide from ribosomal protein L7 on 80 S ribosome by biotinylation. FEBS Lett 287: 121-124

Marion MJ, Marion C (1987) Localization of ribosomal proteins on the surface of mammalian 60S ribosomal subunits by means of immobilized enzymes. Correlation with chemical cross-linking data. Biochem Biophys Res Comm 149: 1077-1083

Mizuta K, Hashimoto T, Otaka E (1992) Yeast ribosomal proteins: XIII. Saccharomyces cerevisiae YL8A gene, interrupted with two introns, encodes a homolog of mammalian L7. Nuc Acids Res 20: 1011-1016

Neumann F, Hemmerich P, Von Mikecz A. Peter H-H, Krawinkel U. (1995) Human ribosomal protein L7 inhibits cell-free translation in reticulocyte lysates and effects the expression of nuclear proteins upon stable transfection into Jurkat T-lymphoma cells. Nuc Acids Res 23: 195-202

von Mikecz A, Neu E, Krawinkel U, Hemmerich P (1999) Human ribosomal protein L7 carries two nucleic acid-binding domains with distinct specificities. Biochem Biophys Res Comm 258: 530-536

Wool IG, Chan Y-L, Gluck A (1995) Structure and evolution of mammalian ribosomal proteins. Biochem Cell Biol 73: 933-947.

A.

Alignment of rat and Arabidopsis L7 proteins. The deduced polypeptides for the genes encoding rat (Rattus norvegicus) and Arabidopsis L7 proteins were aligned using CLUSTAL W. Gaps were introduced to ensure maximum homology. Amino acids that differ from rat L7 are indicated. The shaded residues indicate residues that are strictly conserved (shaded red) or contain very conservative substitutions (V/I/L; Y/F; K/R; shaded gray) in an alignment of eukaryotic L7 sequences including Arabidopsis, potato (Solanum tuberosum), rat, Drosophila and yeast.

B.

Amino acid phylogenetic analysis of L7/L30 r-proteins confirms two distinct types of L7 proteins in plants. A phylogenetic tree was generated by the neighbor-joining method using uncorrected distances in PAUP version 4.0b10 (PPC) based on the amino acid sequence of 9 eukaryotic L7 r-proteins and one archaebacterial L30 protein. Bootstrap values from 500 replicates are indicated. Parsimony bootstrap values for clades supported above the 50% level are indicated above branches, whereas neighbor-joining bootstrap values based on uncorrected distances are indicated below the branches.