The formation of mature mRNAs in vertebrates involves the cleavage and polyadenylation of the pre-mRNA, 10-30 nt downstream of an AAUAAA or AUUAAA signal sequence. The extensive cDNA data now available shows that these hexamers are not strictly conserved. In order to identify variant polyadenylation signals on a large scale, we compared over 8700 human 3Ј untranslated sequences to 157,775 polyadenylated expressed sequence tags (ESTs), used as markers of actual mRNA 3Ј ends. About 5600 EST-supported putative mRNA 3Ј ends were collected and analyzed for significant hexameric sequences. Known polyadenylation signals were found in only 73% of the 3Ј fragments. Ten single-base variants of the AAUAAA sequence were identified with a highly significant occurrence rate, potentially representing 14.9% of the actual polyadenylation signals. Of the mRNAs, 28.6% displayed two or more polyadenylation sites. In these mRNAs, the poly(A) sites proximal to the coding sequence tend to use variant signals more often, while the 3Ј-most site tends to use a canonical signal. The average number of ESTs associated with each signal type suggests that variant signals (including the common AUUAAA) are processed less efficiently than the canonical signal and could therefore be selected for regulatory purposes. However, the position of the site in the untranslated region may also play a role in polyadenylation rate.The 3Ј untranslated regions (UTRs) of eukaryotic mRNAs contain regulatory elements affecting mRNA translation, stability, and transport. Mature 3Ј UTRs are formed by polyadenylation of the pre-mRNA, a coupled reaction involving endonucleolytic cleavage followed by poly(A) synthesis. A significant fraction of mRNAs display multiple polyadenylation sites (Gautheret et al. 1998). The choice of poly(A) sites may influence the stability, translation efficiency, or localization of an mRNA in a tissue-or disease-specific manner (Edwalds-Gilbert et al. 1997). In the mammalian system, effective polyadenylation requires two main sequence components: a highly conserved AAUAAA signal located 10-30 nucleotide 5Ј to the cleavage site and a more variable GU-rich element, 20-40 bases 3Ј of the site (see Proudfoot 1991; Colgan and Manley 1997 for reviews). Although the AAUAAA signal is often considered to be present in 90% of the mRNAs and replaced by a AUUAAA variant in the other 10% (Wahle and Keller 1996; Colgan and Manley 1997), alternate signals are certainly present in a significant fraction of the 3Ј ends (Claverie 1997;Gautheret et al. 1998;Tabaska and Zhang 1999;Graber et al. 1999).The expressed sequence tag (EST) database, dbEST (Boguski et al. 1993), which contains highly redundant partial cDNAs, especially from the 3Ј UTRs, is a rich source of information on mRNA 3Ј ends. Analyzing clustered EST sequences, we previously identified multiple cases of alternate polyadenylation in mRNA (Gautheret et al. 1998). Based on a public EST collection now containing over 1.4 million human sequences, the present work focuses on the region immediatel...

We have used a site-specific cross-linking strategy to identify RNA and protein factors that interact with the 5' splice site region during mammalian pre-mRNA splicing. Two different pre-mRNA substrates were synthesized with a single 32p-labeled 4-thiouridine residue 2 nucleotides upstream of the 5' splice site. Selective photoactivation of the 4-thiouridine residue after incubation of either substrate under splicing conditions in HeLa nuclear extract resulted in cross-links to the U5 snRNA and the U5 snRNP protein p220. These ATP-dependent interactions occur before the first step of splicing. The U5 snRNA cross-links map to a phylogenetically invariant 9-nucleotide loop sequence and do not require Watson-Crick complementarity to the 5' exon. Cross-links of this position in the pre-mRNA to U1, but not to U2, U4, or U6 snRNAs, were also observed. The kinetics of U1 and U5 cross-link formation are similar, both peaking well before reaction intermediates appear.[Key Words: Site-specific cross-linking; pre-mgNA splicing; U5 sngNP; 5' splice site; 4-thiouridine]Received September 8, 1992; revised version accepted October 20, 1992.Noncoding sequences called introns interrupt many eukaryotic genes and must be removed accurately from pre-messenger RNA (pre-mRNA) to yield mature mRNA. This splicing reaction occurs in two steps (for review, see Steitz et al. 1988;Green 1991;Guthrie 1991). The first step is cleavage at the 5' intron-exon junction concomitant with formation of a 2'-5' phosphodiester bond between the 5' end of the intron and a branchpoint nucleotide within the intron to generate a lariat intermediate. In the second step, the free 3' hydroxyl group of the 5' exon attacks the phosphodiester bond at the 3' splice site, resulting in ligation of the exons and release of the intron lariat. This splicing mechanism resembles that of the RNA-catalyzed self-splicing of group II introns found in fungal and plant mitochondria and plant chloroplasts (Guthrie 1991;Sharp 1991).Nuclear pre-mRNA splicing in eukaryotic cells is mediated by a large complex, the spliceosome, which contains five small nuclear RNAs (snRNAs) (U1, U2, U4, US, and U6 snRNAs) and numerous proteins (Reed 1990; for review, see Steitz et al. 1988;and Green 1991). Identification of RNA and protein factors that interact specifically with the pre-mRNA has been crucial to the understanding of the mechanisms of splice site selection and spliceosome assembly. Two snRNA/pre-mRNA intPresent address: Isis Pharmaceuticals, Carlsbad Research Center, Carisbad, Cali|ornia 92008 USA. 2These authors contributed equally to this work.

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The phosphorothioate oligonucleotide T2G4T2 was identflied as an inhibitor of HIV infection in vitro by combinatorial screening of a library of phosphorothioate oligonucleotides that contained all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded tetrameric guanosine-quartet structure. Tetramer formation and the phosphorothioate backbone are essential for antiviral activity. The tetramer binds to the human immunodeficiency virus envelope protein gpl20 at the V3 loop and inhibits both cell-to-cell and virus-to-cell infection. Since functional inhibition of viral infection rather than nucleic acid binding is the selection criterion, the chances are enhanced that a useful compound will be identified.When the SURF strategy was used in a functional screen for an inhibitor of HIV infection, phosphorothioate oligonucleotides with four consecutive guanosines were found to have activity. Of the active compounds, T2G4T2 was chosen for further studies. Physical characterization of this oligonucleotide demonstrated that it formed a tetramer stabilized by G quartets. G-quartet structures are intra-or intermolecular four-stranded helices stabilized by planar Hoogsteen-paired quartets of guanosine (8, 9). The structures are stabilized by monovalent ions bound between two planes ofG quartets and coordinated to the carbonyl oxygens. The G-quartet structure and a phosphorothioate backbone were shown to be required for antiviral activity.Cell culture experiments revealed that the G-quartet structure blocks binding of HIV virions to cells and virus-mediated cell fusion. Virus and cell membrane fusion is initiated by binding of gp120, the major envelope protein of HIV, to its cellular receptor. The mechanism by which membrane attachment occurs is not well understood, but a particular region of gp120, the V3 loop, is essential. The V3 loop maintains a high percentage ofpositively charged amino acids across all HIV strains (10), and the extent of virus-mediated cell fusion and rapid viral replication have been correlated with increased cationic composition of the V3 loop (11). In vitro studies showed that the G-quartet structure bound directly to the V3 loop of gpl20. We hypothesize that the G-quartet structure provides a scaffold to present the phosphorothioate groups in a favorable orientation for binding to the highly cationic V3 loop. MATERIALS AND METHODSOligonucleotide Synthesis. Phosphorothioate oligonucleotides were synthesized by standard protocols (12). For oligonucleotides with randomized positions, the proportions of the individual amidites in a mixture were adjusted until equal amounts of the four dimers were obtained when coupled to dT on controlled-pore glass, as judged by reversed-phase HPLC. Oligonucleotides were purified by reversed-phase HPLC with a gradient of methanol in water. Several purified oligonucleotides were analyzed for base composition by total digestion with nuclease followed by reversed-phase HPLC (Len Cummins, personal communication) and yielded the expected rati...

Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.