The ribotoxin restrictocin recognizes its RNA substrate by selective engagement of active site residues.

Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein-RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.

Electrostatic interactions guide the active site face of a structure-specific ribonuclease to its RNA substrate.

Restrictocin, a member of the alpha-sarcin family of site-specific endoribonucleases, uses electrostatic interactions to bind to the ribosome and to RNA oligonucleotides, including the minimal specific substrate, the sarcin/ricin loop (SRL) of 23S-28S rRNA. Restrictocin binds to the SRL by forming a ground-state E:S complex that is stabilized predominantly by Coulomb interactions and depends on neither the sequence nor structure of the RNA, suggesting a nonspecific complex. The 22 cationic residues of restrictocin are dispersed throughout this protein surface, complicating a priori identification of a Coulomb interacting surface. Structural studies have identified an enzyme-substrate interface, which is expected to overlap with the electrostatic E:S interface. Here, we identified restrictocin residues that contribute to binding in the E:S complex by determining the salt dependence [partial differential log(k 2/ K 1/2)/ partial differential log[KCl]] of cleavage of the minimal SRL substrate for eight point mutants within the protein designed to disrupt contacts in the crystallographically defined interface. Relative to the wild-type salt dependence of -4.1, a subset of the mutants clustering near the active site shows significant changes in salt dependence, with differences of magnitude being >or=0.4. This same subset was identified using calculated salt dependencies for each mutant derived from solutions to the nonlinear Poisson-Boltzmann equation. Our findings support a mechanism in which specific residues on the active site face of restrictocin (primarily K110, K111, and K113) contribute to formation of the E:S complex, thereby positioning the SRL substrate for site-specific cleavage. The same restrictocin residues are expected to facilitate targeting of the SRL on the surface of the ribosome.

The common and the distinctive features of the bulged-G motif based on a 1.04 A resolution RNA structure.

Bulged-G motifs are ubiquitous internal RNA loops that provide specific recognition sites for proteins and RNAs. To establish the common and distinctive features of the motif we determined the structures of three variants and compared them with related structures. The variants are 27-nt mimics of the sarcin/ricin loop (SRL) from Escherichia coli 23S ribosomal RNA that is an essential part of the binding site for elongation factors (EFs). The wild-type SRL has now been determined at 1.04 A resolution, supplementing data obtained before at 1.11 A and allowing the first calculation of coordinate error for an RNA motif. The other two structures, having a viable (C2658U*G2663A) or a lethal mutation (C2658G*G2663C), were determined at 1.75 and 2.25 A resolution, respectively. Comparisons reveal that bulged-G motifs have a common hydration and geometry, with flexible junctions at flanking structural elements. Six conserved nucleotides preserve the fold of the motif; the remaining seven to nine vary in sequence and alter contacts in both grooves. Differences between accessible functional groups of the lethal mutation and those of the viable mutation and wild-type SRL may account for the impaired elongation factor binding to ribosomes with the C2658G*G2663C mutation and may underlie the lethal phenotype.

Differential expression of cysteine dioxygenase 1 in complex karyotype liposarcomas.

Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC) in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs) had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs) (P < 0.001). Location of the tumor was not predictive of the expression level of CDO1 mRNA in any histological subtype of liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs) undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation.

Telomerase suppresses formation of ALT-associated single-stranded telomeric C-circles.

Telomere maintenance is an essential characteristic of cancer cells, most commonly achieved by activation of telomerase. Telomeres can also be maintained by a recombination-based mechanism, alternative lengthening of telomeres (ALT). Cells using ALT are characterized by the presence of ALT-associated promyelocytic leukemia (PML) bodies (APB), long, heterogeneously sized telomeres, extrachromosomal telomeric circular DNA, and elevated telomeric recombination. Consistent with other reports, we found that liposarcomas containing APBs, but lacking telomerase expression, always contained C-rich circles (C-circles), and these C-circles were never present in the absence of APBs, indicating a tight link between these features in ALT cells. However, a rare subgroup of tumors showing evidence of telomere maintenance by both telomerase and ALT did not contain C-circles. To test the hypothesis that telomerase expression disrupts the tight link between APBs and C-circles, we used ALT cell lines that were engineered to express telomerase. Introduction of telomerase activity in these ALT cells resulted in, on average, shorter telomeres with retention of APBs. However, at high passage, the level of C-circles was significantly reduced, which was paralleled by a switch from C-strand overhangs to G-strand overhangs. We propose that by extending critically short telomeres in these cells, telomerase is disrupting a key step in the ALT pathway necessary for production and/or maintenance of C-circles.

Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin.

Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to form a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k2/K1/2 = 1.1 x 10(9) M-1 s-1). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a single-stranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex (E.S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.