Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences

Abstract

Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

FIG. 1

Schematic diagram of the in vitro selection strategy used to identify splicing enhancer sequences. The diagram illustrates the strategy used to construct the dsx-N18 transcription template, transcribe the dsx-N18 pre-mRNA, splice the dsx-N18 substrate in vitro, isolate the functional N18 splicing enhancers, and regenerate the transcription template for subsequent rounds of selection (see Materials and Methods for details). Boxes represent exon sequences, and horizontal lines represent intron sequences (including the branched, excised lariat). Thick boxes represent double-stranded nucleic acids (PCR template, transcription templates, and PCR products), and thin boxes represent single-stranded nucleic acids (DNA oligonucleotides, RNAs, and cDNAs). Solid arrows represent sequential steps in the construction, processing, and regeneration of the DNA and RNA molecules used in the dsx-N18 selection. Unfilled arrows indicate PCR primers (see Materials and Methods for sequences) and their sites of hybridization. In the dsx-N18 construct, the N18 enhancer is positioned at +90 relative to the dsx weak 3′ splice site. The 5′ cap, 5′ splice site, and 3′ splice site in the pre-RNAs are indicated by GG, GU, and AG, respectively. The line connecting the exons indicates the splicing pattern of the female-specific dsx IVS3 minigene upon activation by a splicing enhancer.

FIG. 2

Evolution of the dsx-N18 pool. (A) The dsx-N18 and dsx-(AAG)₆ constructs are shown schematically. Exon 3, intron 3, exon 4, and the enhancer(s) are indicated by E3, IVS3, E4, and N18 or AAG₆, respectively. The 5′ and 3′ splice sites are indicated by GU and AG, respectively. (B) Kinetic analysis showing in vitro splicing assays performed with HeLa cell nuclear extracts and uniformly labeled pre-mRNA splicing substrates comprising the total pool of dsx-N18 pre-mRNAs after various rounds of the selection (rounds 1, 2, 4, and 6 are shown in lanes 4 to 6, 7 to 9, 10 to 12, and 13 to 15, respectively). The negative control pre-mRNA (lanes 1 to 3) is an dsx pre-mRNA lacking an enhancer [dsx(enh⁻)]. The positive control pre-mRNA (lanes 16 to 18) is a dsx pre-mRNA activated by six consecutive copies of a multimerized AAG trinucleotide splicing enhancer (modeled after a synthetic polypurine splicing enhancer in reference 66) that is otherwise isogenic to the dsx-N18 construct. In the kinetic analysis shown, the reaction mixtures were incubated for the number of hours indicated at the top, and positions of the precursors, intermediates, and products of the splicing reaction are indicated to the left and right. The RNAs were analyzed on a 10% denaturing gel in order to resolve the lariat-exon 4 intermediate from the spliced product. (C) Quantitation of the in vitro splicing reactions in panel B. The splicing efficiency (ratio of spliced product to precursor) is calculated from quantitation of individual bands after subtraction of background using a BAS2000 phosphorimager.

FIG. 3

Sequence comparison of individual dsx-N18 enhancers to other naturally occurring splicing enhancer sequences characterized previously by point mutation(s), block substitutions, or deletion mutations. Sequence homology is indicated by boxes. Individual dsx-N18 clone sequences and consensus motifs are designated as in Tables 1 and 2. The ASLV-6U mutant (mut.) shown has no loss of splicing enhancer function compared to the wild type (wt) yet shows an altered pattern of cross-linked factors (60). The third of the six point mutations in the ASLV-6U mutant generates a better match to the class V consensus motif. All other mutants result in at least some loss of splicing enhancer function in vitro or in vivo. Sequence polymorphisms, point mutations, and substitution mutations are underlined. Deletions are indicated by Δ. Due to the large size of some deletions, some sequence information inclusive of the deletion is omitted and indicated by an ellipsis.

FIG. 4

Functional characterization of SC35-dependent enhancers. (A) The dsx pre-mRNA substrates are indicated schematically, with the dsx portion shown in black. Sequences of the different splicing enhancers tested are in capital letters; sequences common to all dsx-N18 clones are in lowercase letters. The dsx-PRE construct is similar but not identical to the dsx-N18 constructs, as it does not contain an inert Sa element. (B) Two dsx-N18 clones that show sequence homology are specifically activated by SC35. The pre-mRNA used in the S100 complementation reactions is indicated above the autoradiogram. The presence of the indicated reaction component in splicing assays and complementation reactions is indicated by a plus sign above the appropriate reaction lane: HeLa cell nuclear extract (NE), HeLa S100 extract complemented with buffer D (S100), or the SR protein indicated by a plus sign. Amounts of SC35 and SF2/ASF used in the complementation assays were 400 and 200 ng, respectively. The dsx-N18 and dsx-PRE pre-mRNAs and spliced products are resolved on a 10% denaturing polyacrylamide gel; their positions are indicated to the right.

FIG. 5

Functional characterization of 9G8-dependent enhancers. (A) The dsx 6-18 pre-mRNA is activated efficiently by 9G8 in S100 complementation assays. The presence of the indicated reaction component in splicing assays and complementation reactions is indicated above the appropriate reaction lane: NE, HeLa cell nuclear extract (lane 1); S100, HeLa S100 extract complemented with buffer D (lane 2). The relative SR protein concentration for each series of SR protein titrations (indicated by a gradient above the lanes) increases by factors of 1.00, 1.50, and 2.25. The actual amounts of SR protein tested in the complementation assays were as follows: 9G8, 67 ng (lane 3), 100 ng (lane 4), and 150 ng (lane 5); SC35, 267 ng (lane 6), 400 ng (lane 7), and 600 ng (lane 8); SF2/ASF, 89 ng (lane 9), 133 ng (lane 10), and 200 ng (lane 11). These amounts were empirically determined to be within the linear complementation range of each SR protein’s specific activity (data not shown). The dsx RNA substrates are indicated schematically, and labeling is as in Fig. 3. The RNA substrates and splicing products were resolved on a 10% denaturing polyacrylamide gel. The reactions were performed in parallel with those in panels B to D. (B) The dsx 6-24 pre-mRNA is activated efficiently by both SC35 and 9G8. (C) The dsx[hβ 117-162] pre-mRNA (57) is activated efficiently by SF2/ASF and by 9G8. (D) The wild-type hβ-globin pre-mRNA (51) is activated efficiently by SC35.

FIG. 6

Binding of SR proteins to individual N18 enhancers. (A) Individual N18 enhancer RNAs were subcloned downstream of a T7 promoter, transcribed in the presence of biotin-21-UTP, and incubated in nuclear extracts by the method of Yeakley et al. (76). The RNA binding proteins were purified by using avidin-agarose and analyzed by Western blotting with MAb 104, which is immunoreactive to a phosphoepitope common to many SR protein family members. Mock refers to a control reaction without RNA included in the assay to determine the background levels of SR recruitment by the avidin-agarose resin in nuclear extracts. The 40-kDa SR protein (present in lanes 2 and 3 but absent in lane 4), the 20-kDa SR protein (present in lane 3 but absent in lanes 2 and 4), and the 35-kDa SR protein (present in lane 4 but absent in lanes 2 and 3) are indicated by asterisks. High-molecular-weight standards (prestained; Bio-Rad) are indicated to the left. (B) The 3-25 enhancer shows good homology to three sequences, dsx PyE (41), CT/CGRT Py (40), and AdML-31 (11), each of which contains a single site-specific label and can cross-link a 20-kDa protein. Solid lines indicate sequence identity, and dashed lines indicate conservative transition changes. Solid asterisks indicate positions of the engineered site-specific labels. The homology among the three sequences that bind SRp20 is CUCKUCY (where K is guanosine or uridine and Y is cytidine or uridine). Triangles integrate the positions of the three site-specific cross-linking experiments.

FIG. 7

Summary of SR protein-specific enhancer sequences. Splicing enhancers (enh.) show specificity in their binding of or activation by individual SR protein family members as characterized by RNA binding studies or S100 biochemical complementation assays. Results for S100 extracts complemented with recombinant SR protein 9G8, SC35, or SF2/ASF (A) and results of studies indicating specific binding of SRp20 (B) are shown. The recruitment assay is the biotinylated RNA affinity technique performed as described by Yeakley et al. (76), and the site-specific cross-linking (SS X-link) refers to the technique developed by Moore and Sharp (48) to incorporate a single ³²P-labeled phosphate into a pre-mRNA substrate. Asterisks indicate positions of single-labeled phosphates positioned within functional pre-mRNAs of dsx PRE (42), hβ-globin SC35 enhancer (57), dsx PyE (41), and AdML (11) or of a site-specifically labeled CT/CGRP Py ribo-oligonucleotide (40). The dsx repeat element (5′ half) shown is an A-type repeat element that is found in three of the six D. melanogaster repeats (31) and all four of the Drosophila virilis repeats (26). Solid lines indicate sequence identity, and dashed lines indicate conservative transitions between splicing enhancer sequences. A putative SRp20 site, based on its sequence identity with the dsx PyE site, is located downstream of an autoregulated 3′ splice site in the SRp20 pre-mRNA (32). K, guanosine or uridine; nd, not determined.