The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation

Abstract

Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter-binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns.

Figure 1. CUGBP2 contacts multiple motifs in the intron upstream from the NI cassette exon.

(A) Schematic of the NI exon (rectangle) and flanking introns (lines) are shown with corresponding nucleotide lengths (numbers, top). RNA substrates (E5-8, E5-10, and E5-15) used for filter binding assays are indicated below. Sequence analyzed by footprinting using primer JBE5-2 (arrow) includes the intron (lower case) and exon (uppercase) region of the 3′ splice site (colon). Shaded sequences represent regions protected from chemical modification by CUGBP2. Individual adenosines in uppercase represent branchpoints determined by primer extension (see below, Figure 4). (B) Nitrocellulose filter binding analysis. ³²P-labeled RNA substrates from (A) were assembled with purified recombinant CUGBP2 protein and separated into protein bound and unbound fractions. Representative graphs for each RNA substrate are shown. Inset: K_d values were calculated as the average of three experiments; ±, standard deviation. (C) RNA footprint analysis. E5-10 RNA was chemically modified with CMCT in the presence or absence of purified CUGBP2 protein. Modified positions were detected by primer extension (lanes +CMCT), in reference to a sequencing ladder generated from plasmid, E5-10 (lanes T,A,C,G). Shaded rectangles at right represent protected regions highlighted in the sequence shown in (A). Primer extension of starting material without modification is shown (lane SM).

Figure 2. The UGUGU core motif and flanking GU dinucleotides are functionally important for CUGBP2 silencing.

(A) Schematic of wild type and mutant versions of the DUPNI splicing reporter. The NI cassette exon (shaded rectangle) and regions of the native flanking introns (shaded lines) were inserted between β-globin exons βE1 and βE2 downstream of a CMV promoter. Nucleotide lengths in base pairs are indicated above and below schematic. Sequence shows an expanded view of the NI 3′ splice site region with mutations m1, m2, and m3 indicated by underscores below the shaded regions. Arrows indicate the location of primers used for RT-PCR amplification of exon included and skipped mRNAs. (B) Splicing reporter expression in C2C12 cells. Splicing reporters with no mutation (wt), with single (m1, m2, m3), or combined (m1,2, m2,3, m1,3, m1,2,3) mutations were expressed with vector backbone control (−) or with pcDNA4/CUGBP2 expression vector (+). The gel panel is a representative polyacrylamide gel image with the top band corresponding to the exon included and the bottom band corresponding the exon skipped mRNA. The bar graph shows the percent exon inclusion as an average of three separate experiments. The change in percent exon inclusion (ΔEI) as a function of CUGBP2 expression is shown below the gel panel. Inset: Western blot analysis was used to verify Xpress-tagged CUGBP2 expression; endogenous hnRNPA1 was a loading control. (C) Experiments were as in (B) except N18TG2 cells were used.

Figure 3. CUGBP2 silencing motifs are functionally transferable.

(A) A 39 nucleotide region containing the m1, m2, and m3 silencing motifs from the NI intron (DIPNIwt) was inserted upstream from DIP13β exon 3 (middle exon) in the SIRT1 splicing reporter context. Expression was driven by the SV40 promoter. Nucleotide lengths in base pairs are indicated. Arrows indicate primers used for RT-PCR amplification; numbers below give nucleotide lengths contributing to PCR products. Individual or combinations of mutations were introduced at CUGBP2 regulatory sites as in Figure 2. Sequence of control region (DIPm93wt) corresponds to 3′ splice site region of constitutive exon DIP13β exon 3. (B) Splicing reporter plasmids were cotransfected in C2C12 cells with vector backbone control (−) or with pcDNA4/CUGBP2 expression vector (+). The graph and ΔEI calculation is as described for Figure 2. (C) Experiments were as in (B) except N18TG2 cells were used.

Figure 4. CUGBP2 blocks branchpoint formation between RNA–protein contact sites.

(A) Schematic of the DUPNIwt pre-mRNA used for in vitro splicing assays. The NI exon and adjacent intron regions are shaded; numbers above schematic indicate nucleotide sizes. Expanded region shows relative positions of CUGBP2 motifs (m1, m2, m3) and branchpoint adenosines (A1, A2, A3) mapped in these experiments. Branch site sequences are shown at right; branchpoint adenosine, asterisk; nucleotides matching the consensus YUNAY (Y, pyrimidine; N, any nucleotide), uppercase; mismatches, lowercase. Alternate registers for CUGBP2 binding are shown schematically in the branch site (BS) perimeter-binding model. (B) Gel panels show primer extension analysis with primer, JBE5-2. Schematic at right illustrates the termination of reverse transcriptase at branchpoint positions in the assay. Branchpoint numbers on gel correspond to positions indicated on sequence in (A). A sequencing ladder is shown for the DUPNIwt plasmid (ladder). For lanes 1–12, splicing reactions containing ATP (+ATP lanes) were incubated for 45 and 60 min with (+) or without (−) 1.6 µM recombinant CUGBP2. Control reactions lacked ATP. For lanes 13–18, 60 min splicing reactions were used.

Figure 5. A motif code for splicing silencing reveals novel endogenous exons that are silenced by CUGBP2.

(A) HEK293T cells were transfected for 36 hours with CUGBP2 protein expression vector (CUGBP2) or vector backbone control (vbb). Exon-included (*) and skipped (**) RT-PCR products are shown after separation on 2% agarose gels. The gene name and exon number are labeled above each panel. The ΔEI values are as follows: SCAMP3_E6, 0±0; MAPT_E2, −5.3±0.6; MAP4_E15, −11.7±3.5; SORBS1_E5, −8±1.7; PPF1BP1_E19, −12.3±2.1; SMARCE1_E4, −21.7±5.5; FOX2_E11, −10±2; CUGBP1_E6, −4.7±0.6; CUGBP2_E6, not determined; ±, standard deviation of three separate experiments. Inset: Western blot confirming CUGBP2 expression (αXpress); total CUGBP2 as detected by 1H2 antibody (αCUGBP2), CUGBP1 and 2 using 3B1 antibody (αCUGBP1,2), and loading control (αhnRNPA1). Black box: RT-PCR detection of CUGBP2 exon 6 skipped mRNA using primers specific for exon 1 and the exon 5/7 junction (jxn). (B) Model depicting the inhibition of branchpoint formation by the binding of CUGBP2 to flanking interaction sites. Below schematic: for exons silenced by CUGBP2, the predicted branch sites between the motifs are shown. Branchpoint adenosine (A*); lowercase letters indicate mismatches to the branch site consensus. Spacing in nucleotides (nt) between the perimeter motifs is shown for all tested exons.

Figure 6. CUGBP2 is autoregulated by silencing from exon 6 branch site perimeters.

(A) Splicing reporter with sequence showing predicted branch sites (A1, A2, A3) and CUGBP2 binding motifs (shaded regions). Primers (arrows) used for RT-PCR (βE1 and βE2) or footprinting analysis (E6) are shown. Mutations in core (CORE) and downstream (DSM) motifs are indicated. (B) Autoregulation of E6 depends upon CUGBP2 binding motifs surrounding the branch site region. Gel panels represent RT-PCR analysis of the wild type or mutant derivatives of the splicing reporter co-expressed with vector backbone control (lanes vbb) or CUGBP2 protein expression vector (lanes CUGBP2) in HEK293T cells. Percent exon inclusion values are shown below gel panels. (C) Footprinting analysis of the predicted branch site region upstream of E6 of CUGBP2 pre-mRNA. Starting material (lane SM), or RNA treated with CMCT in the absence (lane 0) or presence (lanes 3.6, 7.2, 14.4 µM) of CUGBP2 protein. Protected regions are indicated as shaded boxes at right with extended regions of protection (ext) from the CORE or DSM motifs indicated by arrows. (D) Log difference of the band intensities of modified nucleotides in the absence or presence of 14.4 µM CUGBP2. Negative values represent regions protected by CUGBP2. Note that the strength of protection decreases with increasing distance from the primer used for primer extension.