Unusual branch point selection in processing of human growth hormone pre-mRNA.

Abstract

Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream fromn the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.The excision of intervening sequences from nuclear premRNAs has been shown to occur via RNA lariat intermediates (for a review, see reference 39). Primary structural requirements for the formation of lariats in nuclear premRNAs involve sequences at the 5' splice site and the polypyrimidine stretch preceding the 3' splice site (1,15,46). In Saccharomyces cerevisiae, the invariant UACUAAC sequence 20 to 60 nucleotides upstream from the 3' end of the intron was found to be necessary for lariat formation, whereby the third adenosine nucleotide functions as the branch acceptor (12,29,48). The search for a similar sequence in higher eucaryotic pre-mRNAs resulted initially in the proposal of a fairly degenerated consensus sequence (26). From further analyses of branch points of a number of naturally occurring genes and of genes with mutant introns, it became clear that no strict consensus sequence surrounding the branched nucleotide could be defined (40,46,50). However, the adenosine as branch acceptor nucleotide appeared to be conserved. Recently, an adenosine residue was also identified as the branch acceptor in group II introns (53, 57). In addition, there seemed to be a distance constraint for the branch acceptor, which for higher eucaryotic premRNAs was always found within 18 to 40 nucleotides upstream from the 3' splice site (40,46,50).Selection of the branch point poses the problem of positioning the branch acceptor such that its 2' OH group is in a sterically favorable position to attack the phosphodiester bond adjacent to the 5' splice site. Recently, group II introns have been shown to be excised as lariats, and at least some of them are capable of self-splicing in vitro (44,53,57). This implies that the functions required for precise splicing are provided in cis and correlates well with a defined RNA secondary structure in group II introns (23, 33, 52). Moreover, similarities in the splicing mechanisms as well as in the intron boundaries suggest that nuclear and group II introns are evolutionarily related (7). However, the excision of nuclear introns is strongly dependent on trans-acting factors such as small nuclear ribonucleoproteins (snRNPs) and prot...

Mammalian U2 snRNP has a sequence-specific RNA-binding activity.

“…Likewise, an exceptionally efficient branch site, UACUAAC , located 59 nucleotides upstream of the 3' splice site, is inactive in a HeLa cell extract (Ruskin et al 1986). In fact, the distance con straint is so strong that in the absence of an adenosine residue within the 18-to 38-nucleotide range, the branch will form at a cytosine rather than at an adeno sine farther upstream (Hartmuth and Barta 1988).…”

“…Although the above experiments indicate that the branch site can direct U2 snRNP binding, previous studies implicate the 3' splice site/polypyrimidine tract as the major determinant of U2 snRNP binding (Ruskin et al 1985(Ruskin et al , 1988Hartmuth and Barta 1988). The yeast RP51A pre-mRNA provides an ideal system to evaluate the relative importance of these two elements.…”

Crystal structure of a model branchpoint–U2 snRNA duplex containing bulged adenosines

“…Unpaired adenosines are a common feature in RNA structures and are disproportionately represented in certain large folded RNAs such as the 16S ribosomal RNA (Gutell et al+, 1985)+ In the few known RNA tertiary motifs, adenosines are present more often then any of the other 3 nt (Ferre-D'Amare & Doudna, 1999)+ In addition, single bulged adenosines within otherwise duplex RNAs are catalytically important in pre-mRNA splicing and group II self-splicing introns and allow the antigenomic HDV self-cleaving RNA to avoid a kinetic folding trap (Perrotta et al+, 1999)+ In nuclear pre-mRNA splicing, introns are removed by a large RNA-protein machine referred to as the spliceosome+ The spliceosome is made up of five ribonucleoprotein particles (snRNPs) and approximately 100 non-snRNP associated proteins that specifically recognize introns and catalyze their removal through two transesterification reactions (reviewed in Staley & Guthrie, 1998;Burge et al+, 1999)+ Three regions within the intron itself are involved in the transesterification reactions, the 59 splice site, the branchpoint sequence (BPS), and the 39 splice site+ In the first transesterification reaction, an adenosine within the BPS attacks the 59 splice site and in the second reaction the newly freed 59 exon attacks the 39 splice site+ The BPS is highly conserved in the yeast Saccharomyces cerevisiae, with a consensus sequence of UACUAAC, and the branchsite adenosine (underlined) is always used to attack the 59 splice site (Rymond & Rosbash, 1992;Spingola et al+, 1999)+ In other organisms, such as humans, the consensus BPS is less conserved, and either of the two adjacent adenosines can be used to attack the 59 splice site (Query et al+, 1994)+ There are also a few examples of the other 3 nt being used as the nucleophile (Adema et al+, 1988;Hartmuth & Barta, 1988;Dyhr-Mikkelsen & Kjems, 1995;Zabolotny et al+, 1997)+ Specific recognition of the BPS occurs several times during splicing+ The BPS is first recognized by a singlestrand RNA-binding protein referred to as the branchpoint binding protein or splicing factor 1 (Arning et al+, 1996; Berglund et al+, 1997)+ The branchsite adenosine is especially important for this interaction, suggesting that it is recognized even in this early event in the splicing pathway+ The branchpoint binding protein appears to be involved only in early recognition because it is present only in the early stages of spliceosome formation (Rutz & Séraphin, 1999)+ Subsequently, U2 snRNP binds and apparently displaces the branchpoint binding protein to form the BPS-U2 snRNP RNA duplex (Parker et al+, 1987;Wu & Manley, 1989;Zhuang & Weiner, 1989)+ In this base-paired structure, the branchpoint adenosine is unpaired and likely bulged out and available for interactions that will position it for nucleophilic attack …”

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.

“…In addition, analysis of the branchpoints in a number of cellular and viral genes identified a loosely conserved sequence PyNPyUPuAPy, with the adenosine residue usually serving as the site of branchpoint formation 18-40 nucleotides upstream of a 3' splice site (Keller and Noon 1984;Reed and Maniatis 1985;. U2 snRNP is thought to interact with the pre-mRNA at the branchpoint region, most likely through direct base-pairing with the pre-mRNA (Black et al 1985;Konarska and Sharp 1986;Parker et al 1987;Bindereif and Green 1987;Hartmuth and Barta 1988;Wu and Manley 1989;. In addition, a factor termed U2AF (U2 snRNP auxiliary factor) has been reported to be necessary for the binding of U2 snRNP to the branchpoint and for splicing complex assembly (Ruskin et al 19881.…”