Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here we describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing.

Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E. coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide. Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.

We have shown previously that truncation of the human ,3-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence.Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit j3-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.All pre-mRNAs that contain introns have a number of specific sequence elements that are essential for accurate and efficient splicing. These elements appear to be contained within the intron and include the 5' and 3' splice sites, the branch point near the 3' splice site, and the polypyrimidine region. The importance of these sequences has been shown by (i) their high degree of sequence conservation between pre-mRNAs (18), (ii) the occurrence of mutations in each of the elements which severely alter or completely inactivate normal splicing (for recent reviews see references 9, 21, and 27), and (iii) the observation that these sequences specifically bind ribonucleoprotein particles (RNPs) shown to be involved in pre-mRNA splicing (reviewed in reference 16).It is likely that there are additional sequence elements in pre-mRNA involved in the regulation of splicing that have not yet been characterized. We have shown previously that efficient removal of intron 1 of the human ,B-globin premRNA can be dramatically reduced in vitro by truncating the transcript so that only 14 nucleotides of the second exon remain. This transcript is able to undergo the first step in splicing, i.e., cleavage at the 5' splice site and lariat formation, but is unable to efficiently generate the final spliced product or the released lariat intron (7). Similar observations have also been reported by Ruskin and Green (24). Reed and Maniatis (23) reported that different exon sequences can affect the efficiency of splice site selection when tested in an in vitro cis-competition assay. Also, examples of mutations affecting splicing at a distance are the skipping of exon 5 in the DHFR gene in Chinese hamster ovary cells (17) or skipping of exon 2 when transcripts with an altered 5' splice site in intron 2 of the rabbit ,-globin gene are spliced in vitro (1). Since alternatively spliced mRNAs are frequently generated by exon skipping, it seems plausible that this important process is controlled by sequences distant from a given intron, possibly via the interaction of a specific trans-acting factor (for a review of alternative splicing, see reference 15).All these results suggest that spl...

Human P-globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. AU of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps-i.e., cleavage at the 3' splice site and exon ligation-need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2' are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.Recently, several systems for in vitro splicing of eukaryotic mRNA have been developed. The early systems relied either on coupled transcription and splicing of mRNA transcripts in whole cell extracts from HeLa cells (1-3) or on the addition of exogenous precursor mRNA to whole cell extracts from HeLa cells (4) or to nuclear extracts from MOPC-315 cells (5, 6). Progress was slow due to the inefficiency of the splicing extracts and the limited availability of substrate RNA. The recent development of a highly efficient nuclear splicing extract (7,8) and the methods to transcribe large amounts of precursor RNA from DNA templates cloned under the SP6 promoter (9, 10) led to rapid advances in this field.The steps in mRNA splicing, formulated by recent in vitro studies (refs. 11-16; see ref. 17 for a recent review), appear to be as follows: a capped RNA transcript is cleaved at the 5' splice site and, simultaneously, the first nucleotide of the intron attacks an adenosine residue located 20-40 nucleotides (nt) upstream from the 3' splice site, forming a lariat structure. This lariat intermediate is then cleaved at the 3' splice site with concomitant ligation of the 5' exon, thus generating a spliced mRNA and the free intron in the lariat form. The details of the phosphodiester bond formation during splicing have been determined (13) and the participation of U1 ribonucleoprotein (U1-RNP) in the splicing reaction has been convincingly shown (18)(19)(20)(21).In spite of this remarkable progress, many aspects of the splicing reaction are still poorly understood. The mechanisms of splice site selection and alternative splicing are unknown as are the properties of the partially purified splicing enzymes (22). The events that occur during a pronounced initial lag period that is observed in vitro (7) MATERIALS AND METHODS DNA templates were prepared by linearizing the plasmid pSP64HbA6 (7) with restriction enzymes that cleave within the second exon and the first intron of the human...

A novel mutation in the anticodon stem of E. coli tRNA1Tyrsu3+ (G43 to U43) has been characterized. The gene coding for the mutant tRNA, carried by phage phi 80DHA61.3 a derivative of phi 80psu3+su0, produces only 20% of mature suppressor tRNA as compared with phi 80psu3+. Both the mutant tRNA precursor and mature tRNA have an altered conformation. The precursor tRNA coded for by phi 80DHA61.3 is processed by RNase P more slowly than the su3+ precursor and does not form as stable an enzyme-substrate complex as does su3+ precursor. phi 80 DHA61.3 also contains a large deletion which begins in the spacer region between the su3+ gene and the su0- gene, extends through the su0- gene and includes most of the repeated region following the tRNA genes.

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.