Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells.

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Dissecting Monomer-Dimer Equilibrium of an RNase P Protein Provides Insight Into the Synergistic Flexibility of 5' Leader Pre-tRNA Recognition.

Ribonuclease P (RNase P) is a universal RNA-protein endonuclease that catalyzes 5' precursor-tRNA (ptRNA) processing. The RNase P RNA plays the catalytic role in ptRNA processing; however, the RNase P protein is required for catalysis in vivo and interacts with the 5' leader sequence. A single P RNA and a P protein form the functional RNase P holoenzyme yet dimeric forms of bacterial RNase P can interact with non-tRNA substrates and influence bacterial cell growth. Oligomeric forms of the P protein can also occur in vitro and occlude the 5' leader ptRNA binding interface, presenting a challenge in accurately defining the substrate recognition properties. To overcome this, concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima. NMR relaxation (R1, R2), heteronuclear NOE, and diffusion ordered spectroscopy (DOSY) experiments were analyzed, identifying a monomeric species through the determination of the diffusion coefficients (D) and rotational correlation times (τc). Experimental diffusion coefficients and τc values for the predominant monomer (2.17 ± 0.36 * 10-10 m2/s, τ c = 5.3 ns) or dimer (1.87 ± 0.40* 10-10 m2/s, τ c = 9.7 ns) protein assemblies at 45°C correlate well with calculated diffusion coefficients derived from the crystallographic P protein structure (PDB 1NZ0). The identification of a monomeric P protein conformer from relaxation data and chemical shift information enabled us to gain novel insight into the structure of the P protein, highlighting a lack of structural convergence of the N-terminus (residues 1-14) in solution. We propose that the N-terminus of the bacterial P protein is partially disordered and adopts a stable conformation in the presence of RNA. In addition, we have determined the location of the 5' leader RNA in solution and measured the affinity of the 5' leader RNA-P protein interaction. We show that the monomer P protein interacts with RNA at the 5' leader binding cleft that was previously identified using X-ray crystallography. Data support a model where N-terminal protein flexibility is stabilized by holoenzyme formation and helps to accommodate the 5' leader region of ptRNA. Taken together, local structural changes of the P protein and the 5' leader RNA provide a means to obtain optimal substrate alignment and activation of the RNase P holoenzyme.

An exon-biased biophysical approach and NMR spectroscopy define the secondary structure of a conserved helical element within the HOTAIR long non-coding RNA.

HOTAIR is a large, multi-exon spliced non-coding RNA proposed to function as a molecular scaffold and competes with chromatin to bind to histone modification enzymes. Previous sequence analysis and biochemical experiments identified potential conserved regions and characterized the full length HOTAIR secondary structure. Here, we examine the thermodynamic folding properties and structural propensity of the individual exonic regions of HOTAIR using an array of biophysical methods and NMR spectroscopy. We demonstrate that different exons of HOTAIR contain variable degrees of heterogeneity, and identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing unambiguously confirm conserved base pair interactions within the structural element, termed helix 10 of exon 4, located within domain I of human HOTAIR. This combined exon-biased and integrated biophysical approach introduces a new strategy to examine conformational heterogeneity in lncRNAs and emphasizes NMR as a key method to validate base pair interactions and corroborate large RNA secondary structures.

Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine.

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

Computer security for data collection technologies.

Many organizations in the developing world (e.g., NGOs), include digital data collection in their workflow. Data collected can include information that may be considered sensitive, such as medical or socioeconomic data, and which could be affected by computer security attacks or unintentional mishandling. The attitudes and practices of organizations collecting data have implications for confidentiality, availability, and integrity of data. This work, a collaboration between computer security and ICTD researchers, explores security and privacy attitudes, practices, and needs within organizations that use Open Data Kit (ODK), a prominent digital data collection platform. We conduct a detailed threat modeling exercise to inform our view on potential security threats, and then conduct and analyze a survey and interviews with technology experts in these organizations to ground this analysis in real deployment experiences. We then reflect upon our results, drawing lessons for both organizations collecting data and for tool developers.

Comparison of different types of phacoemulsification tips. III. Morphological changes induced after multiple uses in an ex vivo model.

PURPOSE: To assess the ultrastructural morphologic changes in the surface's micro-roughness for single-use and multiple-use phaco tips after multiple phacoemulsification cycles in an ex vivo animal model. SETTING: John A. Moran Eye Center and Utah Nanofab, College of Engineering, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Eight types of phaco tips were studied using an identical experimental protocol. Two tips of each type were used to emulsify porcine cataractous lenses of moderate hardness for 2 minutes. This cycle was then repeated 5 consecutive times for each phaco tip. Scanning electron microscopy and white-light interferometry were used to analyze each tip for potential ultrastructural damage. RESULTS: No significant structural damage was noted on either single- or multiple-use tips after 5 cycles of experimental use. There was a trend toward increased surface micro-roughness at the tip opening after multiple procedures. Most tips had salt crystals on their surface and deposits of organic material composed of carbon and oxygen, which were consistent with porcine tissue that had not been cleaned from the tip surface. CONCLUSIONS: No significant ultrastructural damage was detected after repeated experimental use of phaco tips in a porcine cataract model. There was no significant difference when phaco tips labeled for single use or multiple uses were compared.

NMR resonance assignments of RNase P protein from Thermotoga maritima.

Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5' precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function.

Further Evaluation of Associations Between Reactive and Proactive Aggression and Suicidal Behavior in a Treatment Seeking Sample of Youth.

This study examined associations between reactive and proactive functions of aggression and suicidal behavior in a sample of outpatient treatment seeking youth (n = 111, 60.5% male) ranging from 6 to 17 years of age (Mean age = 10.57 years). Additionally, hope was evaluated as a moderator of these associations. Child reports of measures were used to evaluate associations. When also considering the variance associated with child depressive symptoms and hope, reactive, but not proactive, aggression was uniquely associated with suicidal behavior. Moreover, hope moderated this association, such that reactive aggression was only positively associated with suicidal behavior when levels of hope were low. Findings and their implications for targeting hope with aggressive youth for the prevention of suicidal behavior are discussed.

Structural Roles of Noncoding RNAs in the Heart of Enzymatic Complexes.

Over billions of years of evolution, nature has embraced proteins as the major workhorse molecules of the cell. However, nearly every aspect of metabolism is dependent upon how structured RNAs interact with proteins, ligands, and other nucleic acids. Key processes, including telomere maintenance, RNA processing, and protein synthesis, require large RNAs that assemble into elaborate three-dimensional shapes. These RNAs can (i) act as flexible scaffolds for protein subunits, (ii) participate directly in substrate recognition, and (iii) serve as catalytic components. Here, we juxtapose the near atomic level interactions of three ribonucleoprotein complexes: ribonuclease P (involved in 5' pre-tRNA processing), the spliceosome (responsible for pre-mRNA splicing), and telomerase (an RNA-directed DNA polymerase that extends the ends of chromosomes). The focus of this perspective is profiling the structural and dynamic roles of RNAs at the core of these enzymes, highlighting how large RNAs contribute to molecular recognition and catalysis.

Comparison of different types of phacoemulsification tips. I. Quantitative analysis of elemental composition and tip surface microroughness.

PURPOSE: To evaluate the elemental composition of phacoemulsification tips and their surface roughness in the microscale. SETTING: John A. Moran Eye Center and Utah Nanofab, College of Engineering, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Seven types of phacoemulsification tips were studied. The phaco tips were examined through energy-dispersive x-ray spectroscopy (EDS) and x-ray photoelectron spectroscopy (XPS) for elemental composition. In addition, the roughness of the opening in all tips was assessed through 3-dimensional white-light interferometry. RESULTS: Elemental analysis showed considerable differences in the surface layers between manufacturers. Alcon tips had a thinner oxidized titanium (Ti) layer in their surface. Through XPS, vanadium was not detected in the superficial layers of any tip, but only in deeper levels. The microroughness surface analysis showed comparable results regarding their root-mean-square (RMS) metric. Maximum peak valley distance values varied and appeared to be dependent on the quality of material process rather than the material itself. CONCLUSIONS: Phacoemulsification tips are made of Ti alloys and showed differences between models, especially regarding their composition in the superficial layers. Their opening end roughness showed an overall appropriate RMS value of less than 1.0 µm in all cases. The existence of small defected areas highlights the importance of adequate quality control of these critical surgical instruments. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Comparison of different types of phacoemulsification tips. II. Morphologic alterations induced by multiple steam sterilization cycles with and without use of enzyme detergents.

PURPOSE: To evaluate the alterations in the morphology and elemental composition of reusable phacoemulsification tips after cleaning and sterilization. SETTING: John A. Moran Eye Center, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: For the main experiment, 2 types of reusable phacoemulsification needles were studied. One tip of each type underwent 1, 2, and 3 autoclave sterilizations with the use of detergents followed by thorough rinsing with sterile water between cycles. Another set of tips underwent the same procedure but without rinsing. Subsequently, phaco tips were examined through scanning electron microscopy and energy-dispersive x-ray spectroscopy to assess morphologic changes and surface deposits. In a second experiment, tips of 8 different types (both reusable and single use) underwent 10 sterilization autoclave cycles without detergents. RESULTS: Residues, mostly comprised of carbon-containing material, were found in extensive areas of tips that were sterilization with enzymes and without rinsing. Smaller and fewer residues were found in tips after sterilization with the use of enzymes and thorough rinsing. Tips that underwent autoclave sterilization without detergents had no bulky deposits on their surface; they mostly had thin layers of sodium and chloride or material discoloration. CONCLUSIONS: Rinsing the phaco tips significantly reduced the size and number of residues after use of enzymatic detergents. However, detergent residues were detected on phaco tip surfaces even after thorough rinsing with sterile water. No major noticeable changes were observed in either single-use or reusable phaco tips after 10 cycles of sterilization without detergents. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Evaluation of long-term biocompatibility and capsular bag opacification with a new silicone-polyimide plate-type intraocular lens in the rabbit model.

PURPOSE: To evaluate the long-term biocompatibility and safety of the new SC9 intraocular lens (IOL) after implantation in rabbit eyes, in accordance with the requirements of the International Organization for Standardization 11979-5, Annex G:2006(E). SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Bilateral phacoemulsification was performed on 9 New Zealand white rabbits; 1 eye received the test IOL and the contralateral eye received a commercially available plate-type silicone control IOL (AA4204VL). Two rabbits were humanely killed at 2 months and the remaining at 6 months. After gross examination of the eyes from the posterior Miyake-Apple view, select IOLs were explanted and had surface staining for implant cytology. All globes were then sectioned and processed for complete histopathologic examination. RESULTS: Overall uveal biocompatibility was similar between the test IOLs and control IOLs throughout the study. The mean posterior capsule opacification at 6 months was 0.8 ± 0.27 (SD) in the test group and 4.0 ± 0 in the control group (P = .001, t test: paired 2-sample for means). The mean posterior synechiae formation at 6 months was 1.28 ± 1.25 in the test group and 2.71 ± 0.75 in the control group (P = .01). A histopathologic examination confirmed the relative lack of capsule opacification in the study eyes compared with the control eyes and the absence of untoward inflammatory reaction or toxicity in all eyes. CONCLUSION: The new IOL maintained an open capsular bag secondary to its design, which appeared to prevent overall capsular bag opacification and retain uveal and capsule biocompatibility. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Optic replacement in a novel modular intraocular lens system.

PURPOSE: The purpose of the study is to evaluate the stability and ease of replacement of a new modular intraocular lens (IOL) system consisting of a hydrophobic acrylic base unit and a hydrophobic acrylic optic in the rabbit model. METHODS: Five New Zealand white rabbits were utilized. Bilateral phacoemulsification was performed; one eye received the test IOL (ClarVista Harmoni Modular IOL System) and the other, a commercially available hydrophobic acrylic control IOL (Alcon AcrySof SA60AT). Slit-lamp examinations were performed weekly during postoperative weeks 1 to 6. IOL replacement procedures were performed at weeks 2 and 6 using standard instruments. The rabbits were sacrificed at the end of the study (week 6), with all globes analyzed grossly using the Miyake-Apple technique and sectioned and processed for histopathologic examination. RESULTS: The base component and the optic of the Harmoni IOL system remained stable and centred throughout the clinical follow up. Disengagement of the optic component from the base and replacement of the optic were safely and easily accomplished at 2 and 6 weeks postoperatively. Explantation of the control lens required posterior synechiolysis and capsular bag manipulation due to the position of the loops at the periphery of the bag, thus causing zonular stress. CONCLUSIONS: Replacement of the Harmoni optic was found to be safe and efficient compared with the control IOL, as manipulation of the capsular bag to remove haptic components is not necessary with the modular design. The base component of the modular system remained stable and centred throughout the study.

G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms.

Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions.

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under normal and oxidative stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5 days after treatment. Finally, none of the drugs affected migration. In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.

Treatment of Exudative Age-related Macular Degeneration: Focus on Aflibercept.

A formulation of aflibercept for intravitreal injection (Eylea) is approved for the treatment of patients with exudative age-related macular degeneration (AMD). Aflibercept has a significantly higher affinity for Vascular endothelial growth factor (VEGF)-A compared with other monoclonal anti-VEGF antibodies. In addition to binding all VEGF-A isoforms, aflibercept also blocks other proangiogenic factors such as VEGF-B and placental growth factor. The VIEW 1 and 2 trials showed this drug achieves improved results in patients with exudative AMD similar to those obtained with monthly ranibizumab, using a bimonthly treatment regimen after a loading dose of three intravitreal injections, which translates to less use of healthcare resources. There is a subgroup of patients that present with persistent fluid after the loading dose that could benefit from monthly injections or personalized proactive treatment after the first year. In the second year of treatment, the Treat and Extend patterns can permit even more lengthening of the time between injections. More data are needed to confirm the optimal monitoring and retreatment dosing, to maintain long-term efficacy. Other preliminary data suggest that patients that do not respond to other anti-angiogenics and patients with special pathologies such as polypoidal choroidopathy or retinal angiomatous proliferation can improve upon switching to aflibercept. To date, the safety profile of aflibercept is excellent and is comparable to other anti-angiogenic treatments.

Structural and functional insights into the N-terminus of Schizosaccharomyces pombe Cdc5.

The spliceosome is a dynamic macromolecular machine composed of five small nuclear ribonucleoparticles (snRNPs), the NineTeen Complex (NTC), and other proteins that catalyze the removal of introns mature to form the mature message. The NTC, named after its founding member Saccharomyces cerevisiae Prp19, is a conserved spliceosome subcomplex composed of at least nine proteins. During spliceosome assembly, the transition to an active spliceosome correlates with stable binding of the NTC, although the mechanism of NTC function is not understood. Schizosaccharomyces pombe Cdc5, a core subunit of the NTC, is an essential protein required for pre-mRNA splicing. The highly conserved Cdc5 N-terminus contains two canonical Myb (myeloblastosis) repeats (R1 and R2) and a third domain (D3) that was previously classified as a Myb-like repeat. Although the N-terminus of Cdc5 is required for its function, how R1, R2, and D3 each contribute to functionality is unclear. Using a combination of yeast genetics, structural approaches, and RNA binding assays, we show that R1, R2, and D3 are all required for the function of Cdc5 in cells. We also show that the N-terminus of Cdc5 binds RNA in vitro. Structural and functional analyses of Cdc5-D3 show that, while this domain does not adopt a Myb fold, Cdc5-D3 preferentially binds double-stranded RNA. Our data suggest that the Cdc5 N-terminus interacts with RNA structures proposed to be near the catalytic core of the spliceosome.