In eukaryotes and archaea, uridines in various RNAs. The RNA-guided RNA modification system alters the primary sequence and modulates the function of target RNAs that include rRNAs, snRNAs, tRNAs, and perhaps mRNAs (Yu et al. 1998(Yu et al. , 2005Cavaille et al. 2000;King et al. 2003;Omer et al. 2003). In humans it is currently estimated that >200 2Ј-O-methylations and pseudouridylations are introduced into rRNA and other RNAs by this system (Maden 1990;Bachellerie and Cavaille 1998;Ofengand and Fournier 1998;Vitali et al. 2003).There are two large families of modification guide RNAs found in both eukaryotes and archaea: C/D RNAs that guide 2Ј-O-ribose methylation (Kiss-Laszlo et al. 1996;Omer et al. 2000) and H/ACA RNAs that guide pseudouridylation (Balakin et al. 1996; Ganot et al. 1997a,b;Tang et al. 2002). Both families of guide RNAs function in the context of RNA-protein complexes (RNPs) that include the enzyme responsible for modification (Filipowicz and Pogacic 2002;Terns and Terns 2002). The functional organization of modification guide RNPs, including the mechanism by which the enzyme associates with a guide RNA and the roles of the other essential proteins in the complex, is a subject of great interest. In C/D RNPs the 2Ј-O-methyltransferase, fibrillarin, associates with a guide RNA primarily via a bridge formed by the other proteins in the complex, Nop56/58 and L7Ae (or Nop56, Nop58, and p15.5 in eukaryotes).

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKR WT ) or PKR with a point mutation in each dsRNA-binding motif (PKR RM ). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKR WT samples after PA treatment, but not in the PKR RM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5′-triphosphate enhanced PKR activity compared with the activity with a 5′-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.PKR | snoRNA | metabolic stress | phosphorylation | RNA-binding protein

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3 ′ UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.

IntroductionThis prospective randomized study aimed to evaluate the role of WBRT + SRS compared to SRS alone and to WBRT alone in improvement of overall survival, brain local control and neurologic manifestations.Patients and methodsThe trial included 60 patients with 1 to 3 brain metastases treated at the Radiotherapy Department, National Cancer Institute. 21 patients received WBRT + SRS, 18 patients received SRS alone and 21 patients received WBRT alone.ResultsMedian local control was significantly better for WBRT + SRS compared to SRS alone & WBRT alone (10 vs 6 vs 5 months, respectively, P = 0.04). There was non significant survival benefit for WBRT + SRS compared to SRS alone & WBRT alone. Survival was significantly better for patients with controlled primary tumor who received WBRT + SRS compared to SRS alone & WBRT alone (median survival was 12 vs 5.5 vs 8 months, respectively. P = 0.027). Regardless of the treatment group, median survival and median local control were highly significantly better for single brain site involvement compared to multiple brain sites involvement (P = 0.003 & P = 0.001, respectively), and median brain local control was significantly better for single lesion compared to multiple lesions (P = 0.05).ConclusionsWBRT + SRS is an effective, safe tool in treatment of patients with 1 to 3 brain metastses improving the brain local control, but further studies with larger number of patients is recommended.

Irradiation and temozolomide (TMZ) chemotherapy are the current standard treatments for glioblastoma multiforme (GBM), but they are associated with toxicity and limited efficacy. Recently, these standard therapies have been used to enhance immunotherapy against GBM. Immunotherapy using the anti-CD47 (immune checkpoint inhibitor) treatment has shown promise in treating multiple tumor types, including GBM. The goal of this current work was to test whether irradiation or TMZ chemotherapy could enhance anti-CD47 treatment against GBM. Our results showed that irradiation and TMZ each significantly enhanced anti-CD47-mediated phagocytosis of GBM cells in vitro. Furthermore, mice engrafted with human GBM that received anti-CD47 combined with focal irradiation or TMZ treatment showed a significant increase in the survival rate compared to those that received a single treatment. The tumor growth in mice that received both anti-CD47 and irradiation was significantly less than that of groups that received either anti-CD47 or focal irradiation. The results from this study may support future use of anti-CD47 treatment in combination with irradiation or chemotherapy to enhance the therapeutic efficacy of GBM treatment.

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5–guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.

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