No abstract

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope, branched forms were observed in which 160 nucleotides of RNA from the 5! terminus were not hydrogen bonded to the single-stranded DNA. DNA se uences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17,20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of vira RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.Most eukaryotic mRNAs bear modifications at both termini; their 3' termini have a tract of poly(A) that ranges in length from 30 to 200 bases (1-4), while their 5' termini are typically capped with a methylated guanine joined through a 5'-5' pyrophosphate linkage to a second nucleotide methylated at its 2' position (5, 6). Both types of modifications of eukaryotic mRNA are known to occur after transcription.All adenovirus mRNAs are thought to contain poly(A) tracts at their 3' termini (7) and be capped with a methylated guanine (8, 9). Specific restriction endonuclease cleavage fragments of adenovirus 2 (Ad2) DNA have permitted the mapping of regions of the genome expressed as mRNA and viral proteins during different stages of the lytic cycle (10-12). Little is known about the molecular mechanisms of viral mRNA synthesis. An important aspect of late mRNA synthesis is thought to be the processing and selection of viral mRNAs from the nucleus (18,14). We have purified a late Ad2 hexon mRNA and found evidence providing some insight into the mechanism of synthesis of this mRNA. MATERIALS AND METHODSIsolation of Ad2 DNA and RNA. Polyribosomal RNA was prepared from Ad2-infected cells 32 hr after infection as described by Flint and Sharp (14,15) and selected by chromatography on poly(U)-Sephadex (16).R-Loop Mapping. The R-loop hybridization mixture was essentially that of Thomas et al. (17) (20). The sample was incubated at 57-60°for 2-3 hr. RESULTSAdenovirus late mRNAs begin to appear on polyribosomes about 13 hr after infection and continue to accumulate in the cell throughout the lytic cycle (21). Thus, to fractionate the most abundant late mRNAs, polyribosomes were prepared from cells 32 hr after infection with Ad2 and poly(A)-containing mRNA was selected by chromatography on poly(U)Sephadex columns. These mRNAs were then resolved into different molecular weight fractions by electrophoresis in 2.4-4.0% linear gradient polyacrylamide gels containing a uniform conc...

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder characterized by the loss of acquired motor and language skills, autistic features, and unusual stereotyped movements. RTT is caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Mutations in MECP2 cause a variety of neurodevelopmental disorders including X-linked mental retardation, psychiatric disorders, and some cases of autism. Although MeCP2 was identified as a methylation-dependent transcriptional repressor, transcriptional profiling of RNAs from mice lacking MeCP2 did not reveal significant gene expression changes, suggesting that MeCP2 does not simply function as a global repressor. Changes in expression of a few genes have been observed, but these alterations do not explain the full spectrum of Rett-like phenotypes, raising the possibility that additional MeCP2 functions play a role in pathogenesis. In this study, we show that MeCP2 interacts with the RNA-binding protein Y box-binding protein 1 and regulates splicing of reporter minigenes. Importantly, we found aberrant alternative splicing patterns in a mouse model of RTT. Thus, we uncovered a previously uncharacterized function of MeCP2 that involves regulation of splicing, in addition to its role as a transcriptional repressor.