Dynamic association of RNA-editing enzymes with the nucleolus

© The Company of Biologists Limited 2003ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus. Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.This study was supported by grants from Fundação para a Ciência e Tecnologia,Portugal, and the European Commission (QLG2-CT-2001-01554). This work was also supported by the MRC, a grant from the British Heart Foundation (PG/98086),Ministerio de Ciencia y Tecnologia of Spain (BFI2002-00454) and Fundación Marqués de Valdecilla' of Santander, Spain (A05/02). J.M.P.D. was supported by a long-term fellowship of the European Molecular Biology Organization (EMBO-ALTF 239-2000).info:eu-repo/semantics/publishedVersio

Clastosome: a subtype of nuclear body enriched in 19S and 20S proteasomes, ubiquitin, and protein substrates of proteasome.

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin-proteasome pathway of proteolysis. We coined the term clastosome (from the Greek klastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place

Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus

Phase I, multicenter, open-label study of intravenous VCN-01 oncolytic adenovirus with or without nab-paclitaxel plus gemcitabine in patients with advanced solid tumors

Background VCN-01 is an oncolytic adenovirus (Ad5 based) designed to replicate in cancer cells with dysfunctional RB1 pathway, express hyaluronidase to enhance virus intratumoral spread and facilitate chemotherapy and immune cells extravasation into the tumor. This phase I clinical trial was aimed to find the maximum tolerated dose/recommended phase II dose (RP2D) and dose-limiting toxicity (DLT) of the intravenous delivery of the replication-competent VCN-01 adenovirus in patients with advanced cancer. Methods Part I: patients with advanced refractory solid tumors received one single dose of VCN-01. Parts II and III: patients with pancreatic adenocarcinoma received VCN-01 (only in cycle 1) and nab-paclitaxel plus gemcitabine (VCN-concurrent on day 1 in Part II, and 7days before chemotherapy in Part III). Patients were required to have anti-Ad5 neutralizing antibody (NAbs) titers lower than 1/350 dilution. Pharmacokinetic and pharmacodynamic analyses were performed. Results 26% of the patients initially screened were excluded based on high NAbs levels. Sixteen and 12 patients were enrolled in Part I and II, respectively: RP2D were 1 x10(13) viral particles (vp)/patient (Part I), and 3.3x10(12) vp/patient (Part II). Fourteen patients were included in Part Ill: there were no DLTs and the RP2D was 1 x10(13) vp/patient. Observed DLTs were grade 4 aspartate aminotransferase increase in one patient (Part I, 1x10(13) vp), grade 4 febrile neutropenia in one patient and grade 5 thrombocytopenia plus enterocolitis in another patient (Part II, 1 x10(13) vp). In patients with pancreatic adenocarcinoma overall response rate were 50% (Part II) and 50% (Part III). VCN-01 viral genomes were detected in tumor tissue in five out of six biopsies (day 8). A second viral plasmatic peak and increased hyaluronidase serum levels suggested replication after intravenous injection in all patients. Increased levels of immune biomarkers (interferon- r,soluble lymphocyte activation ne-3, interleukin (IL)-6, IL-10) were found after VCN-01 administration. Conclusions Treatment with VCN-01 is feasible and has an acceptable safety. Encouraging biological and clinical activity was observed when administered in combination with nab-paditaxel plus gemcitabine to patients with pancreatic adenocarcinoma

Access to melanoma drugs in Spain: a cross-sectional survey

BackgroundThe development of highly active drugs has improved the survival of melanoma patients, but elevated drug prices place a significant burden on health care systems. In Spain, the public health care system is transferred to the 17 autonomous communities (AACC). The objective of this study is to describe the situation of drug access for melanoma patients in Spain and how this decentralized system is affecting equity.MethodsFrom July to September 2023, a cross-sectional survey was sent to members of the Spanish Multidisciplinary Melanoma Group (GEM Group). The questionnaire consulted about the real access to new drugs in each hospital. The responses were collected anonymously and analyzed according to several variables, including the AACC.ResultsThe survey was answered by 50 physicians in 15 AACC. No major differences on access between AACC were observed for indications that are reimbursed by the Spanish Health Care System (adjuvant immunotherapy for stage IIIC-IIID and resected stage IV melanoma). Important differences in drug access were observed among AACC and among centers within the same AACC, for most of the EMA indications that are not reimbursed (adjuvant immunotherapy for stages IIB-IIC-IIIA-IIIB) or that are not fully reimbursed (ipilimumab plus nivolumab in advanced stage). Homogeneously, access to adjuvant targeted drugs, TIL therapy and T-VEC, is extremely low or non-existing in all AACC.ConclusionsFor most indications that reimbursement is restricted out of the EMA indication, a great diversity on access was found throughout the different hospitals in Spain, including heterogeneity intra-AACC