Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5 splice sites. We find that a recombinant GST-MS2 protein interferes with 5 splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5 splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.The alternative splicing of pre-messenger RNA is now recognized as a most important generator of protein diversity in mammals. A recent estimate suggests that more than half of the human genes may be alternatively spliced (1). Given that some pre-mRNAs are spliced to yield dozens and potentially thousands of isoforms, the tight control of alternative splicing events is undoubtedly of the highest importance for many cellular and physiological functions. In addition, many human genetic diseases are caused by mutations that affect splice site choice. Thus, procedures that can specifically influence the use of a target splice site will be profitable tools that will help understand the function of protein isoforms, as well as offer novel opportunities to intervene in the correction or the reprogramming of splice site choice for the benefit of human health.Recent efforts aimed at uncovering natural strategies used by the cell to control splice site selection have identified a variety of sequence elements and factors that influence splice site utilization. Specific exonic or intronic sequence elements help to recruit components of the splicing machinery (reviewed in Refs. 2 and 3). In addition, many control elements are bound by factors that prevent the efficient use of adjacent splice sites (4 -20). Whereas a detailed understanding of the molecular mechanisms associated with the activity of such repressors is often lacking, the assembly of a complex that directly impedes recognition of a nearby splicing signal has been described (4, 5). The sequestration of splicing signals into a duplex structure has also been associated with natural examples of splicing repression (21).An artificial approach to control splice site use has been pioneered by Kole and collaborators (reviewed in Ref...

General cytotoxicity was assayed for ceramic (AI20 a, ZrO2/Y2Oa, AIN, B4C, BN, SiC, Si3N 4, TiB, TiC, TiN) diamond and graphite powders, using 3T3 Balb/c permanent cell lines. Neutral red test was carried out in order to establish cell viability. Further investigations were undertaken on human differentiated cells (human umbilical venous endothelial cells): cell behaviour (MTT assay, total cell protein content) and differentiation (immunofluorescence) were studied. In both cases, no cytotoxic effect has been noticed. All the impurities contained at low concentration in these powders do not seem to present any effect. The correlation which has been previously observed between cytotoxicity-cell culture response and blood haemolysis for polymers has not been established here for ceramic powders. We conclude that all the ceramic powders tested here and therefore the corresponding bulk ceramics or ceramic coatings do not induce any cytotoxic effect.

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