Characterization of human RNA splice signals by iterative functional selection of splice sites

Abstract

An iterative in vitro splicing strategy was employed to select for optimal 39 splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 39 end of the randomized insert. Mutating the 39 terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.

Murine serine proteases MASP-1 and MASP-3, components of the lectin pathway activation complex of complement, are encoded by a single structural gene

“…The 3 0 splice site of MASP-3 is preceded by quite extensive polypyrimidine tracts, comprising 29-32 nucleotides with three interrupting purines, whereas the polypyrimidine preceding the competing 3 0 splice site of the MASP-1 serine protease domain exon comprises only 14-18 nucleotides. Similarly, the probable branch site of MASP-3 (about 40 nucleotides upstream of the 3 0 splice site) is pyrimidine-TGAC-pyrimidine, which is a reasonably close fit to the optimal mammalian sequence, 23,24 whereas the corresponding sequence for MASP-1 serine protease domain is purine-TGAC-pyrimidine in rat and mouse.…”

Sodium channels SCN1A, SCN2A and SCN3A in familial autism

“…The substitution of G for the branchpoint site A in intron 6 of SCN1A (TTTTAAT to TTTTAGT) alters the consensus sequence YNY-TRAY and is predicted to reduce splicing efficiency. 26,27 The association of haploinsufficiency of SCN1A with severe seizures 20 supports the possibility that a hypomorphic allele of SCN2A could contribute to autism. However, this mutation did not reduce the efficiency of removal of intron 6 in lymphoblasts.…”

“…Type: P, polymorphic (allele frequency>1%); R, rare (frequencyo1%). Substitution of G for A at the lariat branchpoint is known to reduce splicing efficiency, 26,27 suggesting that this intronic variant could have functional significance. We therefore screened an additional 112 patients and identified 2 more families carrying the same mutation ( Figure 4).…”

Defining a 5??? splice site by functional selection in the presence and absence of U1 snRNA 5??? end

“…Interactions between the 59 splice site of the pre-mRNA and U1, U5, or U6 snRNAs that are supported by experiments+ U1 snRNA is believed to form a transient interaction with the 59 splice site prior to splicing and is replaced by U6 and U5 snRNAs that interact with the same sequences at the time splicing occurs+ Functional selection of 59 splice sites 167 are unknown (Liu et al+, 1998;Sha et al+, 1998;Ismaili et al+, 2001)+ A picture emerges where a given 59 splice site region is recognized through a complex network of interactions that changes during the course of splicing+ We have previously used an iterative functional selection approach to study the recognition of the branch point and 39 splice site (Lund et al+, 2000)+ Here we extend these studies by developing an in vitro strategy for iterative selection of functional 59 splice site sequences from a pool of pre-mRNAs containing randomized inserts+ We provide strong evidence for the hypothesis that factors other than the 59 end of the U1 snRNA determine the shape of the 59 splice site+ Moreover, based on splicing studies of individual pre-mRNAs with variable complementarity to the U1 snRNA, we conclude that stabilizing U1 snRNA binding to the 59 splice site in the natural range increases the competitive strength of a splice site, but concurrently inhibits the assembly of the full spliceosome+…”