Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV. Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination.
The mammalian spliceosome-associated protein, SAP 49, is associated specifically with U2 snRNP and is the most efficiently UV cross-linked protein in the spliceosomal complexes A, B, and C. We show here that SAP 49 cross-links to a region in the pre-mRNA immediately upstream of the branchpoint sequence in the prespliceosomal complex A. In addition to the RNA-binding activity of SAP 49, we show that this protein interacts directly and highly specifically with another U2 snRNP-associated spliceosomal protein, SAP 145. We have isolated a cDNA-encoding SAP 49 and find that it contains two amino-terminal RNA-recognition motifs (RRMs), consistent with the observation that SAP 49 binds directly to pre-mRNA. The remainder of the protein is highly proline-glycine rich (39% proline and 17% glycine). Unexpectedly, the SAP 49-SAP 145 protein-protein interaction requires the amino-terminus of SAP 49 that contains the two RRMs. The observation that SAP 49 and SAP 145 interact directly with both U2 snRNP and the pre-mRNA suggests that this protein complex plays a role in tethering U2 snRNP to the branch site.[Key Words: U2 snRNP; prespliceosome; branchpoint sequence; proline domain; protein-protein interaction] ReceivedThe sequential transesterification reactions that generate spliced pre-mRNA are thought to be carried out by an RNA-catalyzed mechanism, and U2, U5, and U6 small nuclear RNAs (snRNAs) are key spliceosomal RNAs involved in these reactions (for review, see Moore et al. 1993;Newman 1994). U2 snRNA forms an essential base-pairing interaction with the branchpoint sequence (BPS) in the intron (Parker et al. 1987;Wu and Manley 1989;. U6 snRNA is thought to be base paired simultaneously to both the 5' splice site and U2 snRNA; this would position the putative catalytic domain of U6 snRNA near both the BPS and the 5' splice site (Madhani and Guthrie 1992;Sawa and Abelson 1992;Sawa and Shimura 1992;Wassarman and Steitz 1992;Lesser and Guthrie 1993;Kandel-Lewis and S6raphin 1993;Sontheimer and Steitz 1993). Finally, U5 snRNA interacts with exon sequences adjacent to both the 5' and 3' splice sites and may play roles both in specifying the cleavage sites at the splice junctions and in holding the exons together for ligation (Newman andNorman 1991, 1992;Wassarman and Steitz 1992;Wyatt et al. 1992; Cortes et al. 1993, Sontheimer andSteitz 1993).Although a direct role for snRNA-pre-mRNA interactions in catalysis is likely, little is known about how ~Corresponding author. each of these RNA-RNA interactions are first formed, or how they are stabilized sufficiently to carry out their functions. Two observations indicate that additional factors other than the snRNAs are needed to establish and maintain the critical base-pairing interactions. First, the U2, U5, and U6 snRNA-pre-mRNA interactions usually involve only a few nucleotides and therefore, would not be expected to form readily, or be stable, under splicing conditions. Second, in metazoa, the sequence elements in the pre-mRNA that participate in these interactions often dev...
We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5 and 3 splice sites, the branchpoint sequence (BPS), or the exons with 32 P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region ϳ25 nucleotides upstream from the 5 splice site. At the 5 splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5 200, was detected in both the B and C complexes. As observed in yeast cells, U5200 also cross-links to intron/exon sequences at the 3 splice site in the C complex and may play a role in aligning the 5 and 3 exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS 72, BPS 70 , and BPS 56 , which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3 splice site. In this case, a protein, AG 100 , cross-links in the A complex and is replaced by another protein, AG 75 , in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.During pre-mRNA splicing, a series of highly dynamic spliceosomal complexes, consisting of multiple protein and small nuclear RNA (snRNA) components, assemble on pre-mRNA in the order E 3 A 3 B 3 C (for reviews, see references 5, 12, 20, 22, 27, and 31). The sequence elements required for assembly of these complexes are located at the 5Ј and 3Ј splice sites and at the branch site, and exon sequences affect the recognition of both splice sites. In metazoans, all of the elements involved in splicing are weakly conserved. Additional specificity is derived from strict constraints on the locations of these elements relative to one another, which presumably allows for complex networks of RNA-protein, protein-protein, and RNA-RNA interactions between factors bound to each element. Studies of the sequences required for splicing indicate that all of the critical elements are recognized multiple times during the splicing pathway. This proofreading, combined with the vast number of specific interactions established, is most likely the key to achieving high fidelity in the splicing reaction. A detailed understanding of the splicing mechanism requires identifying all of the factors that recognize each of the elements during the different stages of spliceosome assembly and understanding how these factors interact with both the pre-mRNA and one another.A great deal of progress in identifying key recognition factors has come from a combination of genetic and...
The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.Multiple alternative splicing events lead to synthesis from the fibroblast growth factor (FGF) receptor 2 (FGFR-2) gene of a family of receptors differing in defined parts of their extra-and intracellular domains (reviewed in references 19 and 21). In its first described version, FGFR-2 contains an extracellular domain made up of three immunoglobulin-like domains (Ig domains), with a stretch of consecutive acidic residues, the acid box, separating the first two Ig domains. A particularly interesting alternative splice concerns sequences of the mRNA coding for the carboxy-terminal half of the third Ig domain, as this region of the receptor appears to be part of the ligand-binding site. Two alternative exons (K-SAM and BEK) code for this part of FGFR-2 (4,20,29,40). Use of the K-SAM exon results in synthesis of a high-affinity receptor for acidic FGF and keratinocyte growth factor (KGF), while use of the BEK exon yields a high-affinity receptor for acidic FGF and basic FGF (16,29,40). Correct control of the BEK-K-SAM splicing choice appears important, since this choice can influence a cell's response to growth factors that it produces itself as well as to those present in its environment. Consistent with this view, we and others have shown that a given cell line uses predominantly one of the two alternative exons, use of the other exon being sufficiently rare that it cannot be detected in a reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis (4, 29). Thus, epithelial cells express the K-SAM form of FGFR-2 but do not produce KGF, an epithelial cell-specific growth factor, while fibroblasts, which secrete KGF, express the BEK form. The consequences of a "wrong" choice can be disastrous: forced expression of the K-SAM receptor form in fibroblasts producing KGF leads to transformation (30).We are interested in determining the mechanisms involved in discrimination between the BEK and K-SAM exons. Pre-mRNA sequence elements representing potential targets for control of splicing include the 5' and 3' splice sites, the branch point sequence, and the associated polypy...
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