Expand+Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code

MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing

Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs

Identification of motifs that function in the splicing of non-canonical introns

The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests a novel mechanism for intron recognition that compensates for a weakened canonical pre-mRNA splicing motif

A comprehensive computational characterization of conserved mammalian intronic sequences reveals conserved motifs associated with constitutive and alternative splicing

Orthologous mammalian introns contain many highly conserved sequences. Of these sequences, many are likely to represent protein binding sites that are under strong positive selection. In order to identify conserved protein binding sites that are important for splicing, we analyzed the composition of intronic sequences that are conserved between human and six eutherian mammals. We focused on all completely conserved sequences of seven or more nucleotides located in the regions adjacent to splice-junctions. We found that these conserved intronic sequences are enriched in specific motifs, and that many of these motifs are statistically associated with either alternative or constitutive splicing. In validation of our methods, we identified several motifs that are known to play important roles in alternative splicing. In addition, we identified several novel motifs containing GCT that are abundant and are associated with alternative splicing. Furthermore, we demonstrate that, for some of these motifs, conservation is a strong indicator of potential functionality since conserved instances are associated with alternative splicing while nonconserved instances are not. A surprising outcome of this analysis was the identification of a large number of AT-rich motifs that are strongly associated with constitutive splicing. Many of these appear to be novel and may represent conserved intronic splicing enhancers (ISEs). Together these data show that conservation provides important insights into the identification and possible roles of cis-acting intronic sequences important for alternative and constitutive splicing

Binding of U2AF65 to human PY tracts validates the U2AF65 SELEX scoring system

Gel shift of human U2AF65 with human PY tract RNA oligonucleotides. RNA sequences used for binding studies. The gene and intron (IVS) of origin are indicated. The Kvalues are the average of triplicate experiments. Kvalues marked with an asterisk are estimated since the levels of protein required to reach saturation exceed the capacity of the experiment. Linear regression of the observed U2AF65 affinities versus the predicted Sscore.<p><b>Copyright information:</b></p><p>Taken from "Identification of motifs that function in the splicing of non-canonical introns"</p><p>http://genomebiology.com/2008/9/6/R97</p><p>Genome Biology 2008;9(6):R97-R97.</p><p>Published online 12 Jun 2008</p><p>PMCID:PMC2481429.</p><p></p

G-rich and C-rich motifs function combinatorially in LCAT intron 4 splicing

LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. Splicing of the LCAT intron 4 mini-genes (WT, MUT1, MUT3, MUT6, MUT 24 and MUT 25) in HeLa cells. Analysis was performed as in Figure 4. Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.<p><b>Copyright information:</b></p><p>Taken from "Identification of motifs that function in the splicing of non-canonical introns"</p><p>http://genomebiology.com/2008/9/6/R97</p><p>Genome Biology 2008;9(6):R97-R97.</p><p>Published online 12 Jun 2008</p><p>PMCID:PMC2481429.</p><p></p