Generation and characterization of wild-type and mutated L22 expression constructs.

<p>(A) Clusters of basic amino acids likely to be involved in RNA binding were identified within the L22 amino acid sequence and are shown underlined with the basic residues highlighted in bold font. The nine amino-terminal residues previously predicted to be the RNA-binding domain are also highlighted in bold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005306#pone.0005306-ShuNu1" target="_blank">[29]</a>. (B) Location of mutations introduced into L22 coding sequence. Constructs expressing L22 lacking either nine amino-terminal or eight carboxy-terminal residues are designated Δ1–9 and Δ120–128, respectively. Point mutations generated in the basic amino acid clusters illustrated in (A) are shown relative to the wild-type sequence (shown directly below the line) and designated by arrows above and below the line. For m80 and m88, constructs with K to E mutations (and R to D) have been designated m80 and m88 while constructs with K to A mutations have been designated m80A and m88A. (C) N-terminal GFP-L22 fusion constructs, depicted in (B), were transiently transfected into 293T cells followed by analysis of protein lysates for protein expression by immunoblot using anti-GFP antibody. GAPDH served as a control for protein loading.</p

L22 binds EBER-1 and 28S rRNA <i>in vivo</i>.

<p>(A) Expression and biotinylation of BAP-L22 in transiently transfected 293T cells. Transfected cells were lysed and expression of BAP-L22 was verified by immunoblot with anti-L22 antibody (top panel). Successful biotinylation was confirmed by immunoblot with HRP-conjugated streptavidin (bottom panel). Specific capture of biotinylated L22 was demonstrated by immunoblot analysis following incubation of protein lysates with avidin agarose and stringent washing (bound). (B) Specific binding of EBER-1 and 28S rRNA by BAP-L22. 293T cells were transiently co-transfected with either BAP or BAP-L22 and EBERs, EBER-1, or EBER-2 as indicated. 48 hrs post-transfection, cells were UV crosslinked and biotinylated L22 was isolated using avidin agarose, as above. RNA was isolated from both the supernatant and pellet of affinity capture reactions. 2.5 µg RNA from each supernatant sample along with entire RNA sample from each pellet was analyzed by northern hybridization using probes specific for EBER-1, EBER-2, and 28S rRNA, as indicated.</p

Clusters of basic amino acids mediate L22 binding to EBER-1.

<p>(A) Binding of GFP-L22 to stem-loop III of EBER-1 (SL3) was tested in RNA EMSA experiments using increasing amounts of protein lysate (1, 5, and 20 µg) generated from 293T cells transfected with GFP-L22. 5 µg of control lysate expressing only GFP was used to assess nonspecific binding. Each 10 µl binding reaction contained 0.05 pmoles ³²P end-labeled SL3 RNA oligonucleotide. Reactions were electrophoresed on 8% native polyacrylamide gels and visualized by autoradiography. (B) L22 binds specifically to EBER-1. Binding specificity of L22 to SL3 was tested by antibody supershift and by competition with unlabeled oligonucleotides. 5 µg of GFP-L22 protein lysate was used in binding reactions. For antibody supershift experiments, 1 µl of anti-GFP or anti-polyhistidine (nonspecific control) antibody was added to binding reactions. In competition experiments, 10× and 100× unlabeled SL3 or mutated SL3 (mSL3) was added. (C) RNA binding capacity of GFP-L22 containing basic residue mutations or truncation of the amino-terminus (left panel) or with internal point mutations (right panel) was tested in RNA EMSA reactions, as described above. Amounts of each protein lysate used in binding reaction were determined by normalizing the level of expression of each mutated L22 construct to the level of GFP-L22 in 5 µg total protein lysate. Abbreviations used are: FP = free probe, NS = nonspecific, E = endogenous, GFP-L22 = all specific shifts generated with wild-type or mutated GFP-L22 proteins, SS = supershift.</p

Mutation of residues 80–93 alters the subcellular localization of L22 and prevents incorporation into ribosomes.

<p>(A) The subcellular localization of mutated GFP-L22 proteins was analyzed by fluorescence microscopy following transient expression in 293T cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005306#pone-0005306-g005" target="_blank">Fig. 5A</a>. (B) Incorporation of m88 into ribosomes was analyzed by sucrose density gradient analysis of extracts generated from 293T cells engineered to stably express m88, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005306#pone-0005306-g005" target="_blank">Fig. 5B</a>. Migration of molecular weight standards (in kDa) is indicated to the left of the blots. Following detection of L22 (15 kDa) and m88 (43 kDa) with anti-L22 antibody, the blot was stripped of antibody and reprobed with anti-GFP antibody to confirm that the 43 kDa bands present in fractions 1–3 were in fact GFP-tagged L22.</p

GFP-L22 is localized to nucleoli and incorporated into ribosomes.

<p>(A) The subcellular localization of wild-type L22 fusion proteins and control proteins was analyzed by fluorescence microscopy following expression in 293T and HeLa cells. For transient expression, cells grown on coverslips were transfected with 2 µg of the indicated expression construct, fixed after 48 hrs and visualized using a Zeiss Axiovert inverted fluorescence microscope. HeLa-L22 cells stably express GFP-L22. BAP-L22 expression was visualized in transiently transfected HeLa cells following staining with Alexa Fluor 488-conjugated streptavidin. Fibrillarin served as an endogenous nucleolar marker and was detected in HeLa cells using anti-fibrillarin antibody and Alexa-conjugated secondary antibody. All coverslips were mounted in Vectashield plus DAPI. Bar equals 10 µm. (B) Nuclear (N) and cytoplasmic (C) fractions from untransfected and GFP-L22 transfected 293T cells were analyzed by immunblot using anti-L22 and anti-GAPDH antibodies. Extract from the indicated number of cells was analyzed. Following detection of L22, blots were stripped of antibody and reprobed for GAPDH which served as a control for cytoplasmic contamination of nuclear extracts. (C) Localization of endogenous L22 and GFP-L22 in HeLa-L22 cells was assessed by sucrose density gradient analysis. Ribosome-containing lysates were separated on a 10–50% w/v sucrose gradient and 0.5 ml fractions were collected from the top of the gradient. The protein and RNA content of each fraction was analyzed by western blot and agarose gel electrophoresis, respectively. Total RNA was visualized by ethidium bromide. Polysome profiles were recorded during fraction collection at 260 nm. The ribosomal subunit composition of each peak is indicated along with fraction numbers corresponding to the first and last fraction collected (1 and 12) as well as the start of collection of the 40S (fraction 6) and 60S (fraction 9) peaks.</p

Mutation of residues 80–93 eliminates binding of L22 to multiple RNA substrates.

<p>The capacity of mutated L22 proteins to bind to EBER-1 (A) and 28S rRNA (B) was determined by specific capture of proteins on biotinylated RNAs immobilized on streptavidin magnetic beads. Protein lysates were generated from transiently transfected 293T cells and normalized for expression relative to wild-type GFP-L22. Total protein was incubated with 100 pmoles of biotinylated RNA and complexes were captured on streptavidin magnetic bead columns. Column flow-thru and eluate were subjected to SDS-PAGE and analyzed for GFP-L22 proteins by immunoblot using anti-GFP antibody. Protein lysate from cells transfected with GFP alone was used as a control for nonspecific binding to beads or RNA.</p