A combinatorial code for splicing silencing: UAGG and GGGG motifs - PubMed (original) (raw)

A combinatorial code for splicing silencing: UAGG and GGGG motifs

Kyoungha Han et al. PLoS Biol. 2005 May.

Abstract

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

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Figures

Figure 1

Figure 1. Exonic UAGG and 5′ Splice Site GGGG Motifs Are Required in Combination for Silencing of the CI Cassette Exon

(A) A GGGG splicing silencer motif at the 5′ splice site. Top: Sequence of the 5′ splice site region (5′ to 3′) with exonic (uppercase) and intronic (lowercase) nucleotides. Numbering is relative to the first nucleotide of the intron. Arrowhead indicates 5′ splice site. A predicted SRp40 motif overlying the last seven bases of the exon is indicated. Engineered mutations and names of splicing reporters are indicated immediately below the affected nucleotides. Effect of mutations on the pattern of splicing is shown in a 5′ to 3′ arrangement (gel panel and graph). All splicing reporter plasmids have a three-exon structure in which CI is the middle exon (in the schematic, vertical bars indicate exons and horizontal lines indicate introns). Splicing reporter plasmids were expressed in vivo in mouse C2C12 cells, and splicing patterns assayed by radiolabeled RT-PCR of cellular RNA harvested from the cells. PCR primers are specific for the flanking exons. Results of multiple experiments are shown graphically as the average percent of exon included in product (y-axis) for each splicing reporter construct (x-axis). (B) Analysis of ESE motifs. An exonic UAGG splicing silencer motif overlaps an ASF/SF2 motif. Sequence of the CI exon (5′ to 3′) is shown, with engineered mutations (underscored) and names of splicing reporters indicated immediately below the affected nucleotides (bold). Numbering is relative to the first nucleotide of the exon. Predicted ESE motifs for ASF/SF2 and SC35 are highlighted above the exonic sequence as indicated in brackets. The UAGG motif required for silencing (boxed) is indicated below the overlapping ASF/SF2 motif (asterisk). Effect of mutations on the in vivo pattern of splicing is shown in a 5′ to 3′ arrangement (gel panel and graph). Error bars in (A) and (B) represent standard deviations.

Figure 2

Figure 2. Effect of Number and Position of CI Cassette Exon Splicing Silencer Motifs

Splicing reporters were constructed with variations in the number and position of UAGG and/or GGGG motifs. Three sets of schematics (boxed at center) illustrate the CI cassette exon and adjacent 5′ splice site region with positions of exonic UAGG (black vertical bars) and 5′ splice site GGGG (grey vertical stripe) motifs. Splicing reporter names are indicated at left. Vertical arrowhead indicates 5′ splice site. Each splicing reporter was generated by site-directed mutagenesis from parent plasmid wt0. Natural UAGG positions 51 and 93 represent the starting position of the motif relative to the first base of the exon. Engineered UAGG positions 11, 76, and 100 are also indicated (see schematic in center box at top). Sequence changes of the mutations are underscored: 11, GUGG→

UA

GG; 51, UAGG→

AU

GG; 76, CCAG→

UAG

G; 93, UAGG→

GU

GG; 100, UCCAA→U

AGGC

. Representative splicing patterns in PC12 cells (left gel panels) and C2C12 cells (right gel panels) are shown together with average percent exon inclusion values. The correlation between motif pattern and strength of splicing silencing is summarized (bottom). Exon-included (double arrowheads) and exon-skipped (single arrowheads) products are indicated.

Figure 3

Figure 3. Identification and Functional Roles of Protein Factors That Bind to GGGG and UAGG Motifs

(A) Detection of protein binding to the 5′ splice site GGGG motif by UV crosslinking in HeLa nuclear extract. Wild-type (cs1 and 3h1) and mutant (cs3 and 3h3) RNA substrates were internally labeled at guanosine nucleotides; mutations are underscored. Pattern of UV crosslinking is shown following RNase digestion and SDS-PAGE (lanes 1–4). Immunoprecipitation reactions (lanes 5–11) contained the 3h1 substrate together with antibody specific for hnRNP F or H/H′; control samples contained preimmune rabbit serum. Gel panel shows the pellet (P), supernatant (S), and input (I) of the immunoprecipitation reactions following SDS-PAGE. The positions of hnRNP H/H′ and F (arrowheads) and protein molecular weight standards (in kilodaltons) are indicated. The hnRNP F and H/H′ antibodies were a gift of C. Milcarek. (B) UV crosslinking of exonic position 93 UAGG motif in HeLa nuclear extract. RNA substrates were prepared with a single radiolabeled nucleotide as indicated by the asterisk; sequences are shown (bottom). The wild-type (wt3) and mutant (mt3) substrates are identical except for the underscored mutation. The A1winner substrate corresponds to the high-affinity hnRNP A1 binding sequence previously identified by SELEX. The position of hnRNP A1 is indicated (arrowhead). Monoclonal antibody 9H10 was a gift of G. Dreyfuss. (C) Exon inclusion is enhanced by co-expression of hnRNP F or H. Gel panel shows splicing pattern resulting from co-transfection of wild-type (wt) or mutant (5m2) splicing reporter with hnRNP F or H expression plasmid; splicing reporters are identical to those shown in Figure 1A. Control samples were transfected with empty vector; grey wedge indicates two levels (4 and 6 μg) of protein expression plasmid. Arrowhead indicates 5′ splice site. For immunoblot verification of transfected protein expression (bottom), nuclear extracts from transfected cells were separated by SDS-PAGE, transferred to nylon membrane, and developed with an antibody specific for the Xpress tag at the N-terminus of each pcDNA–protein sample. Raw percent exon inclusion values are shown below gel image. (D) Silencing effect of hnRNP A1 requires the intact 5′ splice site GGGG motif and full-length downstream intron. Structures of chimeric splicing reporters are shown in which the CI cassette exon and intron flanks were introduced into an unrelated splicing reporter containing sequences from the GABAA receptor γ2 transcript: rGγCI-wt0 (both introns truncated), -up (full-length upstream intron, truncated downstream intron), and -dn (truncated upstream intron, full-length downstream intron). Numbers above indicate length of each intron segment in nucleotides. Arrowhead indicates 5′ splice site. The splicing reporters rGγCI-dn5m2 and -dn5m4 contain the full-length downstream intron with 5′ splice site mutations of Figure 1A. Gel panel shows splicing pattern resulting from co-transfection of splicing reporter with hnRNP A1 expression plasmid or vector control. Immunoblot verification of transfected protein expression (bottom) is as described in (C).

Figure 4

Figure 4. Computational Analysis of UAGG and GGGG Motif Patterns Reveals Association with Exon Skipping Genome-Wide

At the top is a flow chart for the computational analysis used to illustrate the procedure used to identify human exons with and without the CI cassette silencer motif pattern (≥1 exonic UAGG and a 5′-splice-site-proximal GGGG), followed by the determination of the percentage of confirmed skipped exons in each group. The reciprocal pattern (≥1 exonic GGGG and a 5′ splice site UAGG) and related variants were analyzed for comparison as indicated in the graph and table. The graph (middle) shows exons with the motif pattern on the left and the remaining exons without the pattern (w/o) on the right; x-axis, 5′ splice site motif; y-axis, percent confirmed skipped exons; z-axis, exonic motif. Confirmed skipped exons were defined as those skipping events supported by 20 or more individual cDNA and/or EST entries. Exonic motifs were allowed at any position within the exon, but not overlapping the splice sites, and the 5′ splice site motif was restricted to bases 3–10 of the intron. Only exons of 250 nucleotides or fewer were considered. The table (bottom) shows, for each motif pattern, the percentage of confirmed skipped exons within that group (as shown in the graph) and the number of exons in the group (in parentheses). The CI cassette silencer motif pattern is boxed.

Figure 5

Figure 5. Genome-Wide Identification of Exons with UAGG and GGGG Silencing Motifs

A database of 96,089 orthologous human and mouse exon pairs was searched for TAGG located anywhere in the exon and GGGG in bases 3–10 of the intron. Venn diagrams indicate the number of exons containing either or both sequence motifs in the human subset and the mouse subset of the database. The number of exons (19) in which UAGG and GGGG silencer motifs are conserved in orthologous human and mouse exons is also shown (intersection). The motif patterns are shown in the context of the exon (uppercase) and 5′ splice site region (lowercase) for 12 examples from the intersection dataset (human sequences are shown). Colon indicates 5′ splice site. The conserved TAGG and GGGG motifs are highlighted in red to illustrate variations in their positions. Gene name (HUGO ID) and exon number within the gene are indicated at far right. For one uncharacterized transcript, the GenBank accession is given instead (NM_018469_8).

Figure 6

Figure 6. RT-PCR Confirmation of Exon Skipping Patterns in Human Tissues

Eleven orthologous exons (≤250 nucleotides in length) were selected from the analysis of Figure 5 for RT-PCR analysis in a panel of eight human tissues. These exons are derived from the intersection dataset, in which conserved TAGG and GGGG motifs are present in combination in the human and mouse orthologous exons. Additional cDNA and EST evidence for these skipping events are summarized in Table 1. Specific primer pairs were designed for each test exon to amplify the exon-included (double arrowhead) and exon-skipped (single arrowhead) products by RT-PCR. Each gel panel shows the products of reactions for a single test exon resolved on agarose gels in the arrangement given in the inset. Gene name, exon number, and Ensembl number (in parentheses) are provided above each gel panel. The far left and far right lanes of each gel panel contain DNA molecular weight markers.

Figure 7

Figure 7. Analysis of Splicing Patterns in Mouse Tissues for Variations in the Number of Exonic UAGGs

Splicing patterns were determined by radiolabeled RT-PCR for selected mouse exons. Control reactions include β-actin exon 2 and HNRPH1 exon 8, which were selected because they lack the silencing motifs studied (“0 TAGG, 0 GGGG exons”). HNRPH1 exon 5 and HNRPH3 exon 3 are representative of the one TAGG plus GGGG motif pattern (“1 TAGG + GGGG exons”). Hp1bp3 exon 2, GRIN1 CI cassette exon, and NCOA2 exon 13 are examples of tissue-specific exon skipping associated with the two TAGG plus GGGG motif pattern (“2 TAGG + GGGG exons”). MEN1 exon 8 is also shown. Each gel panel shows splicing patterns tested in RNA samples from mouse heart and brain tissue and mouse C2C12 cells. Gene name, exon number, and Ensembl gene ID (in parentheses) are provided above each gel panel. Curly brackets point to the average percent exon inclusion and standard deviation for each set of serial dilutions; raw values are given immediately below each lane. Sequence alignments (bottom left) of the corresponding human and mouse orthologs illustrate the patterns of silencer motifs (orange). Bold indicates an additional exonic GGGG motif.

Figure 8

Figure 8. Analysis of UAGG and GGGG Motif Pattern in a Heterologous Context and Effects of hnRNP A1 and H Co-Expression

At the top is a schematic of the heterologous splicing reporter SIRT1 (pZW8) that contains exon 6 of the human SIRT1 gene and flanking intron sequences as described previously [17]. The intron/exon lengths (in nucleotides) are as follows: exon 1, 308; intron 1, 340; exon 2, 95; intron 2, 287; and exon 3, 436. The silencing motif pattern was introduced sequentially into the middle exon and adjacent 5′ splice site region as highlighted in red. GGGG mutations were introduced by site-directed mutagenesis at positions 6–9 or 8–11 of the second intron. Exonic UAGG motifs were introduced into the middle exon by replacing a HindIII-KpnI restriction fragment

AAGCTT

TCGAATTC

GGTACC

, with

AAGCTT

GTTAGGTA TA

GG TACC

(restriction sites are underscored) as described [17]. The percent exon inclusion values (average of three repeats) were determined from co-expression assays with vector backbone (vbb) or with a 1:4 ratio of hnRNP A1 or H expression plasmid (same as experiment of Figure 3). Below are shown splicing assays following expression in C2C12 cells in the absence and presence of hnRNP A1 (“A1” lanes) or hnRNP H (“H” lanes) protein expression vector. Control reactions contained vector backbone plasmid (“−” lanes). Exon-included (double arrowheads) and exon-skipped (single arrowheads) products are indicated.

Figure 9

Figure 9. Exons Identified by Bioinformatics to Contain UAGG and GGGG Motif Patterns: Analysis of Splicing Patterns in a Heterologous Context and Effects of hnRNP Co-Expression

Exons were cloned from mouse genomic DNA with 12 nucleotides of flanking intron sequence on each side. Each fragment was inserted into the _SIRT1_a context between the NdeI and XbaI restriction sites (Test Exon). _SIRT1_a is identical to SIRT1 except for the NdeI and XbaI sites located 12 nucleotides upstream and downstream of the middle exon, respectively. The segment between the NdeI and XbaI restriction sites represents the region of _SIRT1_a replaced by test exons. ESS19a is the same as ESS19 except for the presence of the indicated restriction sites. Test exons included the CI cassette exon of rat GRIN1 (GRIN1_CI), exon 8 of MEN1 (MEN1_8), and exon 2 of Hp1bp3 (Hp1bp3_2). Splicing reporters were expressed in C2C12 cells in the presence of vector backbone (vbb), or with hnRNPA1 or hnRNPH protein expression vector at a ratio of 1:4. Exon-included (double arrowheads) and exon-skipped (single arrowheads) mRNA products were quantified from the gel shown, and used to calculate the percent exon included values (top right).

Figure 10

Figure 10. Computational Analysis of Exonic UAGG Motifs and Exon Skipping Patterns Genome-Wide

Computational searches were performed to identify exons with two or more UAGGs and to determine the association of confirmed exon skipping events with this group. Exons with a single UAGG were analyzed for comparison. The following constraints were applied: (1) exon lengths of 250 bases or fewer and (2) both UAGG motifs conserved in sequence and position in the orthologous mouse exons. The graph illustrates the percentage of confirmed exon skipping events associated with one UAGG or two or more UAGGs (blue bars), or with the remaining exons lacking these motifs (red bars). The list of 16 human exons identified with two or more UAGGs is shown with the Ensembl ID, exon number, 5′ splice site sequence, and gene name. It is not unexpected to find exon 19 of the glutamate NMDA receptor GRIN1 and exon 13 of NCOA2, which have a 5′-splice-site-proximal GGGG, since the sequence of the 5′ splice site was not specified in the search.

Figure 11

Figure 11. Model for Differential Regulation of the CI Cassette Exon by the Interplay of hnRNP A1 and H and a Ternary Motif Pattern

At the top is a schematic of intron/exon structure and prominent splicing patterns observed in the forebrain (top) and hindbrain (bottom) of rat brain. Below is a summary of splicing regulatory motifs functionally defined in this study depicted on an expanded version of the GRIN1 CI cassette exon (yellow). ESEs are indicated above the exon. Nucleotides complementary to U1 small nuclear RNA and the interaction of the positive regulator NAPOR/CUGBP2 with the downstream intron are indicated (‡; as determined in [26]). UAGG and GGGG splicing silencing motifs defined in this study are highlighted in red. The working model for splicing silencing, based on the results shown here, proposes that the CI cassette is a strong exon silenced by a combination of two exonic UAGG motifs and a 5′-splice-site-proximal GGGG. HnRNP A1 mediates silencing and hnRNP H mediates anti-silencing via these RNA signals.

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