The receptor for advanced glycation endproducts (RAGE) mediates responses to cell danger and stress. When bound by its many ligands (which include advanced glycation endproducts, certain members of the S100/calgranulin family, extracellular high-mobility group box 1, the integrin Mac-1, amyloid beta-peptide and fibrils), RAGE activates programs responsible for acute and chronic inflammation. RAGE is therefore also involved in cancer progression, diabetes, atherosclerosis, and Alzheimer's disease. RAGE has several isoforms deriving from alternative splicing, including a soluble form called endogenous secretory RAGE (esRAGE). We show here that most soluble RAGE, either produced by cell lines or present in human blood, is not recognized by an anti-esRAGE antibody. Cells transfected with the cDNA for full-length RAGE, and thus not expressing esRAGE, produce a form of soluble RAGE, cleaved RAGE (cRAGE) that derives from proteolytic cleavage of the membrane-bound molecules and acts as a decoy receptor. By screening chemical inhibitors and genetically modified mouse embryonic fibroblasts (MEFs), we identify the sheddase ADAM10 as a membrane protease responsible for RAGE cleavage. Binding of its ligand HMGB1 promotes RAGE shedding. Our data do not disprove the interpretation that high levels of soluble forms of RAGE protect against chronic inflammation, but rather suggest that they correlate with high levels of ongoing inflammation.

Several proteins have been identified that protect Drosophila telomeres from fusion events. They include UbcD1, HP1, HOAP, the components of the Mre11-Rad50-Nbs (MRN) complex, the ATM kinase, and the putative transcription factor Woc. Of these proteins, only HOAP has been shown to localize specifically at telomeres. Here we show that the modigliani gene encodes a protein (Moi) that is enriched only at telomeres, colocalizes and physically interacts with HOAP, and is required to prevent telomeric fusions. Moi is encoded by the bicistronic CG31241 locus. This locus produces a single transcript that contains 2 ORFs that specify different essential functions. One of these ORFs encodes the 20-kDa Moi protein. The other encodes a 60-kDa protein homologous to RNA methyltransferases that is not required for telomere protection (Drosophila Tat-like). Moi and HOAP share several properties with the components of shelterin, the protein complex that protects human telomeres. HOAP and Moi are not evolutionarily conserved unlike the other proteins implicated in Drosophila telomere protection. Similarly, none of the shelterin subunits is conserved in Drosophila, while most human nonshelterin proteins have Drosophila homologues. This suggests that the HOAP-Moi complex, we name ''terminin,'' plays a specific role in the DNA sequenceindependent assembly of Drosophila telomeres. We speculate that this complex is functionally analogous to shelterin, which binds chromosome ends in a sequence-dependent manner.HP1 ͉ MRN complex ͉ telomeric fusions ͉ telomeric proteins

Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. Drosophila telomeres are protected by terminin, a complex that includes the HOAP (Heterochromatin Protein 1/origin recognition complex-associated protein) and Moi (Modigliani) proteins and shares the properties of human shelterin. Here we show that Verrocchio (Ver), an oligonucleotide/oligosaccharide-binding (OB) fold-containing protein related to Rpa2/Stn1, interacts physically with HOAP and Moi, is enriched only at telomeres, and prevents telomere fusion. These results indicate that Ver is a new terminin component; we speculate that, concomitant with telomerase loss, Drosophila evolved terminin to bind chromosome ends independently of the DNA sequence.Supplemental material is available at http://www.genesdev.org.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

Helicases are ubiquitous enzymes that unwind or remodel single or double-stranded nucleic acids, and that participate in a vast array of metabolic pathways. The ATP-dependent DEXH-box RNA/DNA helicase MLE was first identified as a core member of the chromatin remodeling MSL complex, responsible for dosage compensation in Drosophila males. Although this complex does not assemble in females, MLE is present. Given the multiplicity of functions attributed to its mammalian ortholog RNA helicase A, we have carried out an analysis for the purpose of determining whether MLE displays the same diversity. We have identified a number of different proteins that associate with MLE, implicating its role in specific pathways. We have documented this association in selected examples that include the spliceosome complex, Helicases are involved in all cellular transactions related to nucleic acid function and metabolism in prokaryotes and eukaryotes. These enzymes participate in transcription, RNA splicing, the stability of transcripts, and translation initiation, as well as in DNA-replication, repair, and recombination. Helicases use the energy produced by the hydrolysis of nucleotide triphosphates to catalyze the unwinding of DNA, RNA, and RNA:DNA hybrid molecules. They are also involved in protein displacement from RNA, RNA clamping, strand annealing, and RNA structure conversion (1). Eukaryotic helicases can be divided into two superfamilies, SF1 and SF2, which include three and nine families, respectively. Members of the different families can be distinguished on the basis of whether they use the hydrolysis of ATP or some other nucleotide triphosphates for energy release, whether they unwind DNA or RNA, and whether they require a 3Ј overhang or a 5Ј overhang to carry out their unwinding functions (2, 3).The Drosophila helicase maleless (MLE) 1 is a subunit of the MSL (male-specific lethal) complex that is responsible for dosage compensation -the regulatory mechanism involved in equalizing the levels of X chromosome-linked gene products between the sexes. In addition to MLE, the MSL complex contains an ubiquitin ligase (MSL2), a histone acetyl transferase (MOF), two structural proteins (MSL1 and MSL3), and one of two long non-coding RNAs (roX1 and roX2) that are necessary for its assembly and targeting. MLE is an ATP-dependent DEXH-box RNA/DNA helicase that in vitro prefers hybrid RNA:DNA or double-stranded RNA substrates with a short 3Ј overhang (4). MLE exhibits some similarity to the ATPases present in complexes that remodel chromatin by altering the positioning or the architectural relationship between histone octamers and DNA. In contrast to MLE, none of these enzymatic subunits have been shown to possess double-stranded nucleic acid unwinding activity. The absence or loss of function of MLE leads to failures in assembly and targeting of the MSL complex to its numerous sites of action along the X chromosome in males, although MSL1 and MSL2 are present at a few "high affinity" or "entry" sites (5-10). Recent biochemical ev...

In Drosophila, dosage compensation is mediated by the MSL complex, which binds numerous sites on the X chromosome in males and enhances the transcriptional rate of a substantial number of X-linked genes. We have determined that topoisomerase II (Topo II) is enriched on dosage compensated genes, to which it is recruited by association with the MSL complex, in excess of the amount that is present on autosomal genes with similar transcription levels. Using a plasmid model, we show that Topo II is required for proper dosage compensation and that compensated chromatin is topologically different from non-compensated chromatin. This difference, which is not the result of the enhanced transcription level due of X-linked genes and which represents a structural modification intrinsic to the DNA of compensated chromatin, requires the function of Topo II. Our results suggest that Topo II is an integral part of the mechanistic basis of dosage compensation.

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