Recognition of the 3 splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3 splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3 splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA. These studies, combined with an analysis of extant high resolution x-ray structures of protein⅐RNA complexes, suggest a model whereby U2AF65 bends the pre-mRNA to juxtapose reactive functionalities of the pre-mRNA substrate and organize these structures for subsequent spliceosome assembly.Removal of non-coding intron sequences from pre-mRNAs 1 in eukaryotes involves two sequential transesterifications. In the first step, the branch point adenosine within the intron carries out a nucleophilic displacement at the 5Ј splice site, producing the 5Ј exon and lariat intermediate. The liberated 5Ј exon attacks the 3Ј splice site to yield ligated exon and lariat intron products. Both reactions are catalyzed by the spliceosome: a large (ϳ60 S) ribonucleoprotein assembly. The spliceosome consists of the U1, U2, and U4/U5/U6 snRNPs (small nuclear ribonucleoprotein particles), each containing a unique snRNA and associated proteins as well as non-snRNP splicing factors (1-3). Assembly of the spliceosome on the pre-mRNA proceeds through the formation of several complexes and is directed by conserved sequences at the 5Ј and 3Ј splice sites as well as other sequences in the pre-mRNA; recognition of the 3Ј splice site is closely coupled to recognition of the proximal branch region and polypyrimidine tract within the intron. Regulation of this assembly process results in differential splice site usage, and the resulting patterns of alternative splicing are a major source of proteome diversity in higher eukaryotes (4, 5).Commitment of a pre-mRNA to the splicing pathway involves the ATP-independent formation of the early or commitment complex on the pre-mRNA substrate (1-3). In mammals, this complex includes U1 snRNP, tightly associated with the 5Ј splice site, as well as non-snRNP protein factors. These proteins include the heterodimer U2AF, containing large (U2AF65) and small (U2AF35) subunits, which binds to the polypyrimidine tract and 3Ј splice site, the branch-binding protein SF1, and members of the SR protein family (6 -11). Following the formation of the commitment complex, U2 snRNP is recruited to the pre-mRNA in an ATP-dependent process. This association involves the formation of a duplex between U2 snRNA and the pre-mRNA branch region, which bulges out the branch adenosine, specifying it as the nucleophile fo...
We have synthesized a novel thiol reagent, 2-[(methylsulfonyl)thio]ethyl [N-(N,N-dimethylamino)ethyl]carbamate (MTSAC), that contains a carbamate functional group as well as a (positively charged) terminal amino group. The carbamate C-N bond isomerizes on a millisecond time scale and significantly alters the three-dimensional shape of the reagent. The behavior of this reagent was contrasted with that of the commonly used thiol reagent, [(methylsulfonyl)thio]ethylamine MTSEA [Akabas, M. H., & Karlin, A. (1995) Biochemistry 34, 12496-12500], with respect to its effect on single-channel currents passing through modified gramicidin channels. While both reagents decreased single-channel currents, the MTSAC-treated channels also showed a pattern of steps in the current recordings on the time scale of the carbamate bond isomerization. Moreover, the pattern and size of these steps were sensitive to the location of the thiol-reactive site in relation to the channel entrance. Thus, MTSAC may prove useful as a reagent for establishing the proximity to the pore in studies of ion channel proteins of unknown structure.
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