RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.

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Background:The R21C mutation in cardiac troponin I (cTnI) prevents PKA-mediated phosphorylation of serines 23 and 24 of cTnI in vivo. Results: Myofilament function is uncoupled from the intracellular [Ca 2ϩ ] and delays muscle relaxation. Conclusion: Long term ablation of cTnI phosphorylation leads to hypertrophy, diastolic dysfunction, and dysautonomia in mice. Significance: Restoration of phosphorylated cTnI may prevent hypertrophic cardiomyopathy and diastolic dysfunction.The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca 2؉ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca 2؉ -transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca 2؉ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca 2؉ content. This abnormal Ca 2؉ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na ؉ -Ca 2؉ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.Inherited as an autosomal dominant disease, familial hypertrophic cardiomyopathy (HCM) 2 is the most common genetic disorder of the heart (1). This clinical syndrome is characterized by an increase in left ventricular mass, diastolic dysfunction, and dysautonomia and carries a high incidence of sudden cardiac death (2). In many HCM cases, cardiac contractile dysfunction is attributed to inherited sarcomeric gene mutations, which can clinically present with variable penetrance, even within the same family pedigree (3,4). Within the context of the sarcomere, it is well established that mutations in cardiac troponin (cTn) alter t...

The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-doublestranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2=OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. IMPORTANCE RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication.Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.

Hepatitis delta virus (HDV) is a unique human pathogen that increases the severity of liver disease in those infected with its helper virus, hepatitis B virus (HBV). The ϳ1,680-nucleotide (nt) single-stranded circular RNA genome assumes a characteristic unbranched rodlike structure in which ϳ70% of the nucleotides form Watson-Crick base pairs (1, 2). Genome replication occurs via a double-rolling-circle mechanism in which host RNA polymerase is redirected to synthesize genomic and antigenomic HDV RNAs, as well as the mRNA for hepatitis delta antigen (HDAg), the sole viral protein (reviewed in references 3 and 4). HDAg forms RNA-protein complexes (RNPs) with both genomic and antigenomic HDV RNAs, and these complexes play essential roles in this unique replication process (5-11). HDAg has been shown to transport HDV RNA to the nucleus, where replication occurs (12). In addition, HDAg interacts with RNA polymerase II (Pol II) (13,14); this interaction may recruit the polymerase to bound HDV RNA and has been proposed to effect changes in the polymerase that permit RNA-directed transcription (14-17). RNPs formed by HDAg and HDV RNA are also packaged into virions (18, 19); these complexes include a modified form of HDAg that interferes with RNA synthesis (20-24). Understanding how HDV RNPs function depends on knowing their structural features, which remains a critical goal.Several studies have attempted to identify regions of HDAg that directly contribute to binding HDV RNA and forming HDV RNPs, but a consistent picture has not yet emerged. In a widely cited model, two arginine-rich motifs (ARM I and ARM II) in the middle region of the protein are thought to form a bipartite RNA binding domain (8). This model is based largely on two in vitro binding studies using bacterially expressed HDAg fusion proteins. One of these studies showed that only fusion proteins containing the middle region of the protein, which includes the ARMs, could bind HDV RNA (25); no RNA binding activity was associated with the N-terminal 78 amino acids (aa). The other study showed loss of in vitro RNA binding activity when either of the ARMs, particularly ARM I, was disrupted by replacement of two basic residues with other amino acids (8). Both reports relied heavily on RNAprotein blots (Northwestern blots), which depend on the ability to remove bound detergent and properly refold proteins following electrophoresis and blotting. In contrast, other in vitro studies implicated the amino-terminal domain of HDAg in binding HDV . However, these analyses were limited by the use of small segments of the protein; Poisson et al. used small peptide fragments (28), and Huang and Wu and Wang et al. employed an N-terminal 88-aa region that bound equally well to HDV and non-HDV RNAs (26,27).Analyses of HDAg-HDV RNA interactions in cells have also yielded conflicting interpretations. Wang et al. (11) found that mutation of ARM I severely diminished the ability of HDAg to package the RNA into secreted particles, a result that could suggest a role for ARM I in ...