Autophagy, the degradation of cytoplasmic components, is an evolutionarily conserved homeostatic process involved in environmental adaptation, lifespan determination and tumour development. The tumor suppressor Beclin1 is part of the PI(3) kinase class III (PI(3)KC3) lipid-kinase complex that induces autophagy. The autophagic activity of the Beclin1-PI(3)KC3 complex, however, is suppressed by Bcl-2. Here, we report the identification of a novel coiled-coil UV irradiation resistance-associated gene (UVRAG) as a positive regulator of the Beclin1-PI(3)KC3 complex. UVRAG, a tumour suppressor candidate that is monoallelically mutated at high frequency in human colon cancers, associates with the Beclin1-Bcl-2-PI(3)KC3 multiprotein complex, where UVRAG and Beclin1 interdependently induce autophagy. UVRAG-mediated activation of the Beclin1-PI(3)KC3 complex promotes autophagy and also suppresses the proliferation and tumorigenicity of human colon cancer cells. These results identify UVRAG as an essential component of the Beclin1-PI(3)KC3 lipid kinase complex that is an important signalling checkpoint for autophagy and tumour-cell growth.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.

The 5'-cap-containing leader sequence of the most abundant 19S and 16S mRNAs of simian virus 40 (SV40) was previously mapped between 0.67 and 0.76 map units. We now find that the two late mRNA species contain multiple 5' ends. Eight different RNase T2-resistant cap structures were identified: m7GpppmAmpU (47%); m7GpppmAmpUmpU (l9%o, m7GpppmAmpC (16% ). m7GpppmAmpCmpA (5%); mGpppmAmpG J6%); miGpppGmpC (3%); m7GpppmAm-GmpA (2%), m GpppGmpCmpG (2%). Capped Ti oligonucleotides of 19S and 16S mRNAs have been isolated by two different procedures: (i) chromatography on a DEAE-cellulose column followed by paper electrophoresis and (ii) two-dimensional electrophoresis/homochromatography. Cap structures of the isolated 5' oligonucleotides were identified. Each of the major caps was found to be associated with a few different 5' oligonucleotides, implying a vast heterogeneity at the termini of SV40 late mRNAs. The results suggest that on SV40 DNA, RNA polymerase II has a repertoire of initiation points. In most of the cases, initiation takes place with adenosine triphosphate followed by a pyrimidine. Alternatively, transcription may start at one specific point but a unique mechanism of processing generates heferogeneous populations of termini with a common 5' adenosine triphosphate. The majority of messenger RNAs in eukaryotic cells, as well as mRNAs of many viruses infecting them, are blocked at their 5'-termini by a cap structure (7-methylguanosine joined through a 5'-5' pyrophosphate bridge to the penultimate nucleotide of the transcribed chain) (1). Capping of mRNA in eukaryotic cells occurs posttranscriptionally (2-4) but is tightly coupled to initiation of transcription (5, 6). The 5'-cap structures are important for ribosome binding, mRNA translation, and protection against nucleolytic degradation (7).During the lytic cycle of simian virus 40 (SV40), after the onset of DNA synthesis, the late region is transcribed into virus-specific messengers that fall into the size classes of 19S and 16S (8, 9). From studies carried out during the past year, it has become evident that, similar to adenovirus-2 (10-13), SV40 late mRNAs are transcribed from noncontiguous sequences of DNA (14-19). The 5' ends of late SV40 messengers were mapped at 0.72 I 0.02 map units (18,(20)(21)(22)(23). Recently, the 5' termini of nascent RNA chains derived from SV40 transcriptional complexes were mapped by electron microscope analysis at coordinate 0.67 + 0.02 (24). Analysis of nuclease Pl-resistant cap structures of SV40 late mRNAs showed that the two late mRNA species possess similar 5' termini (m7GpppmAm) in which the penultimate nucleotide is dimethylated adenosine (25,26).Hoping to identify the promoter for SV40 late transcription, we have examined the 5'-capped oligonucleotides of the late mRNAs. As presented below, we have found that, in contrast to adenovirus-2, in which all the late messenger RNAs (at least 12 different species) have one 5'-terminal oligonucleotide (27)(28)(29) Enzymatic Treatment and Product Analysis. Enzyme ...

No abstract

No abstract