The L7Ae RNA binding motif is a multifunctional domain required for the ribosome-dependent Sec incorporation activity of Sec insertion sequence binding protein 2

Abstract

The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA([Ser]Sec) and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.

FIG. 1.

Multiple sequence alignment of the SBP2 L7Ae RNA binding domain. SBP2 amino acid sequences from rat, chicken, puffer fish, and fly were aligned. Residues shaded in black are identical, and residues shaded in gray are conservative substitutions. Residues mutated to penta-alanine are indicated above the corresponding positions in SBP2. Sequences were generated using MultAlin, and conserved residues were shaded using Boxshade.

FIG. 2.

Ribosome binding analysis of CTSBP2 penta-alanine mutant proteins. (A) Wild-type and mutant forms of CTSBP2 were translated and [³⁵S]Met labeled in rabbit reticulocyte lysate and resolved by 12% SDS-PAGE. (B) The proteins described in the legend for panel A were incubated with purified rat ribosomes and centrifuged through a 20% sucrose cushion. Equal portions (5%) of the supernatant (S) and pellet (P) were resolved by SDS-PAGE and analyzed by phosphorimaging. The luciferase construct was translated and tested as a negative control. (C) The results presented in panel B were quantitated as the percentage of protein in the pellet and normalized relative to the level of pelleting observed for wild-type CTSBP2. The amount of ribosome binding obtained with in vitro-translated hnRNP F (33% pelleting) was considered background and subtracted. The data shown are the averages ± standard errors of results from at least three independent experiments.

FIG. 3.

SECIS binding analysis of CTSBP2 penta-alanine mutant proteins. (A) Diagram of the PHGPx SECIS element. The 203-nucleotide PHGPx SECIS element was utilized to assay SECIS binding. The SECIS core motif is boxed to indicate the SBP2 binding site. (B) [³⁵S]Met-labeled wild-type and mutant forms (indicated by the regions corresponding to the mutations) of CTSBP2 were incubated with 20 fmol of the wild-type ³²P-labeled PHGPx SECIS element, and the complexes were resolved on a 4% nondenaturing gel. The asterisk indicates the position of a SECIS-specific complex. Unsupplemented reticulocyte lysate (retic alone) was tested as a negative control to account for nonspecific binding. (C) The results presented in panel B were quantitated as the percentage of shifted probe, corrected for nonspecific binding, and normalized relative to the level of shift observed for wild-type CTSBP2. The data shown are the averages ± standard errors of results from at least three independent experiments.

FIG. 4.

Sec incorporation activity of CTSBP2 penta-alanine mutant proteins. The mutant proteins (indicated by the regions corresponding to the mutations) represented in Fig. 1 were added to a reticulocyte lysate assay mixture containing luciferase mRNA with a Sec codon at position 258 and a wild-type PHGPx SECIS element inserted in the 3′ UTR. After normalization for protein expression, the luciferase activity of each mutant protein was normalized relative to the luciferase activity of wild-type CTSBP2, set at 100%. For each SBP2 mutant protein, the average luciferase activity is shown, together with the SECIS and ribosome binding data from Fig. 2 and 3.

FIG. 5.

Analysis of CTSBP2 forms with pairwise subdivisions of penta-alanine mutations. The RFQDR^647-651 segment and the flanking segments subjected to penta-alanine mutation (LLKEL^641-645 and RRLVL^664-668) were each analyzed in four mutant proteins (indicated by the corresponding mutation) with pairwise substitutions of alanine. Each mutant protein harboring a pairwise substitution, along with the corresponding penta-alanine mutant protein (indicated by the region corresponding to the mutation), was assayed for ribosome binding, SECIS binding, and Sec incorporation activity. The data are the means (± standard errors) of results from at least three independent experiments.

FIG. 6.

Structural modeling of SBP2. (A) The cocrystal structure of the 15.5-kDa snRNA binding protein bound to its cognate K-turn U4 RNA (Protein Data Bank coordinates 1E7K) was used to predict the positions of the following regions within CTSBP2: RFQDR^647-651, DGAQD^746-750, G669 (yellow), LVL^666-668, RR⁶⁶⁴, KALGR^725-729, LLKEL^641-645, FHKMV^752-756, LNKAV^731-735, and PVSIV^736-740. (B) Space-filled model of the structure presented in panel A, highlighting the auxiliary and core functional regions within the L7Ae domain as noted. The annotation of the structure was performed using MacPyMOL. (C) Alignment of the L7Ae motifs from the following K-turn binding proteins: Saccharomyces cerevisiae (Sc) rpL30, the human (Hs) 15.5-kDa protein, and the SBP2 sequences in Fig. 1. Residues shaded in black are identical, and residues shaded in gray are conservative substitutions. A secondary structure prediction for SBP2 (developed using PredictProtein) (26) is shown above the sequences.

FIG. 7.

SECIS elements modulate the SBP2-ribosome interaction. (A) [³⁵S]Met-labeled wild-type CTSBP2 and the RFQDR^647-651 and G669R mutant forms were incubated with increasing amounts (0, 0.25, 0.5, 1.0, and 2.0 pmol) of a wild-type (WT) PHGPx SECIS element or a mutant element lacking the core motif (ΔAUGA) and analyzed for ribosome binding as described in the legend to Fig. 2B. S, supernatant; P, pellet. (B) The data obtained as described for panel A were quantitated by phosphorimaging, normalized for nonspecific binding, and graphed as the percentage of ribosome binding relative to that for the no-SECIS reaction mixture. (C) Four picomoles of recombinant CTXH was subjected to UV cross-linking to 20 fmol of the PHGPx SECIS element. Following cross-linking, reaction mixtures were incubated with excess salt-washed purified ribosomes (10 pmol) and spun as described in the legend to Fig. 2. Equal portions of the supernatant and pellet (9%) were resolved on an SDS-PAGE gel and analyzed by phosphorimaging (top panel) or by Western blot analysis (bottom panel). A reaction mixture containing a mutant SECIS element (ΔAUGA) was used as a negative control.