A novel subgenomic murine leukemia virus RNA transcript results from alternative splicing

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD') within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD' was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD' without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.     

Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.     

Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.     

Two forms of human cytoplasmic arginyl-tRNA synthetase produced from two translation initiations by a single mRNA

Human cytoplasmic arginyl-tRNA synthetase (ArgRS) is a component of a macromolecular complex consisting of at least nine tRNA synthetases and three auxiliary proteins. In mammalian cells, ArgRS is present as a free protein as well as a component of the complex. Via an alignment of ArgRSs from different vertebrates, the genes encoding full-length human cytoplasmic ArgRS and an N-terminal 72-amino acid deletion mutant (hcArgRS and DeltaNhcArgRS, respectively) were subcloned and expressed in Escherichia coli. The two ArgRS products were expressed as a soluble protein in E. coli. The level of production of DeltaNhcArgRS in E. coli and its specific activity were higher than those for hcArgRS. By Western blot analysis, using an antibody against the purified DeltaNhcArgRS, the two forms of ArgRS were detected in three human cell types. The 5'-end cDNA sequence, as confirmed by 5'RACE (5'-rapid amplification of cDNA ends), contained three start codons. Through mutation of the three codons, the two human cytoplasmic ArgRSs were found to be produced in different amounts, indicating that they resulted from two different translation initiation events. Here we show evidence that two forms of human cytoplasmic ArgRS were produced from two translational initiations by a single mRNA.     

Complete in vitro reconstitution of adeno-associated virus DNA replication requires the minichromosome maintenance complex proteins

Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.     

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.     

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

A G-rich element forms a G-quadruplex and regulates BACE1 mRNA alternative splicing

β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the APP to the amyloid β-protein (Aβ), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5' splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in Aβ production from APP, suggesting new possibilities for therapeutic approaches to Alzheimer's disease.     

Functions of Lsm proteins in mRNA degradation and splicing

Recent results have identified a family of Lsm (Like Sm) proteins that are related to the Sm protein family. Seven Lsm proteins form a complex, which interacts with the U6 snRNA and functions in splicing. In addition, a different complex of Lsm proteins interacts with cytoplasmic mRNA and promotes its turnover. These diverse functions of Lsm proteins suggest that they are important modulators of RNA biogenesis and function.