Structuring of the 3′ Splice Site by U2AF65

Abstract

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...

Strict 3′ splice site sequence requirements for U2 snRNP recruitment after U2AF binding underlie a genetic defect leading to autoimmune disease

“…Another example is stabilizing interactions mediated by arginineserine-rich (RS) domains, which support protein-protein interactions as well as interactions between proteins harboring these domains and double-stranded RNA regions (e.g., those resulting from base pairing between U2 snRNA and the branch site region) (Wu and Maniatis 1993;Green 2004 2006;. Furthermore, the arrangement of protein and RNA contacts in the initial complex has been proposed to enforce a unique RNA structure in the 39 splice site that may be critical to trigger subsequent events in the splicing process (Kent et al 2003). In addition, proofreading activities exist that ensure proper assembly of splicing factors in bona fide splice site signals, e.g., allowing the assembly of U2AF on uridine-rich sequences only if followed by a 39 splice site AG dinucleotide (Soares et al 2006).…”

FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35

“…U2AF65 binds specifically to the Py tract via its C-terminal RNA-binding domain, which consists of three RNA recognition motifs (RRMs) (Ruskin et al 1988;Zamore et al 1992; for review, see Krämer 1996). U2AF65 stabilizes the interaction of U2 snRNP with the BP by contacting the branch region through its N-terminal arginine-and serine-rich (RS) domain, promoting base pair interactions between U2 snRNA and the BP (Lee et al 1993;Gaur et al 1995;Valcarcel et al 1996;Kent et al 2003). In previous models it was suggested that U2AF65 function is based on interactions between RRM3 and SF1/ mBBP, or the U2 snRNP component SAP155, respectively (Berglund et al 1997;Gozani et al 1998).…”

Evolution of a tissue-specific splicing network

“…LS additionally cooperates with the branch point adenosine-binding SF1 through interactions in its C-terminal pseudo-RNA recognition motif (RRM) (Kent et al 2003;Selenko et al 2003). Following these contacts, the 39 end of the intron is then competent for interaction with U2 snRNP.…”