DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose.

This corrects the article DOI: 10.1038/cdd.2015.26.

DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose.

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions.

Intrahelical pseudoknots and interhelical associations mediated by mispaired human minisatellite DNA sequences in vitro.

The human minisatellite arrays, 33.6 and 33.15, consist of tandem reiterations of a 37-nucleotide (nt) and a 16-nt repeat unit sequence, respectively, both of which contain a majority of purine bases on one strand. Knot-like tertiary structures, which mapped to the cloned arrays, were observed by electron microscopy (EM) in homoduplex molecules produced by denaturation and reannealing in vitro. They result from a primary hybridization between misaligned repeat units of the array, forming a slipped-strand structure with staggered single-stranded DNA loops, followed by a secondary hybridization between repeat units in the two loops. Depending on the relative alignment of the loops when they hybridize, a particular form of intrahelical pseudoknot is produced. Theta-shaped, figure-of-eight, and bow-shaped structures were the most common conformational isomers observed in homoduplexes flattened into two dimensions during EM preparation. At the site of a bow-shaped structure, a conformation-dependent bend of approximately 60 degrees between the flanking DNA segments is induced; the other conformations generally do not deflect the line of the main DNA axis. Paired loops, similar to the bow-shaped structure, were apically situated in some supercoiled plasmids containing the 33.6 array. Both plasmids formed intermolecular associations, consisting of two (or more) homoduplex molecules held together at or immediately adjacent to a nexus which mapped to the minisatellite sequences. These associations might arise either by interhelical hybridization between arrays or by knot-like structures interfering with branch migration of chi-form Holliday junctions.

Multiple Harvey-ras genes in the bovine genome.

Three different Harvey-ras (Ha-ras) genes have been identified in the bovine genome by screening a lambda phage-bovine DNA library with the ras gene of the Harvey murine sarcoma virus. The genes have been characterized by hybridization and heteroduplex analysis with both the viral and human Ha-ras genes. Based on heteroduplex mapping, the putative direction of transcription and the approximate position of the coding regions of the bovine genes were established. Two genes, bovine Ha-ras 1 and 2, both show an arrangement of exons and introns typical of c-Ha-ras genes. However, they are distinct entities as they have different nucleotide sequences and physical maps, and heteroduplexes between them contain a region of non-homology upstream of exon 1. The third gene, c-Ha-ras 3, does not contain introns and is a pseudogene, analogous to human c-Ha-ras 2. The nucleotide sequence of both Ha-ras 1 and Ha-ras 2 has been determined and shows that Ha-ras 1 encodes a bona fide ras protein, whereas Ha-ras 2 has diverged considerably from a recognizably functional sequence.

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.

R-loop mapping of RNA transcripts from bovine papillomavirus type 4.

RNA isolated from bovine oesophageal warts was hybridised to BPV-4 genomic DNA cloned in a plasmid vector. In R-loop preparations, six major classes of transcripts were mapped. Class I, due to an unspliced transcript encompassing the E4/E5 ORF region, is most common. In Class II the E4/E5 region is spliced at its 5' end to the E6 ORF region, and the RNAs appear to have different transcription start points in the E6 ORF. Some Class I R-loops may represent shorter Class II-type transcripts not hybridised to the E6 region. Transcripts that form Class III R-loops have not been previously described for BPV-4 and contain the E4/E5 ORF exon spliced at its 3' end to the LI ORF. In Class IV, transcripts map to the 3' end of the EI exon, artificially truncated by the Bam HI site used for cloning the BPV-4 genome, and are spliced to the 5' end of the exon containing the E4/E5 ORF. Class V transcripts are ambiguously located in the cloned BPV-4 genome, and could be derived from the EI or LI ORF. The former case may represent the remainder of the transcript from Class IV R-loops. The rare Class VI R-loops are due to the L2 ORF spliced at its 3' end to the LI ORF.