Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1
© Palaniswamy et al; licensee BioMed Central Ltd. 2010
Received: 9 October 2009
Accepted: 30 April 2010
Published: 30 April 2010
Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited.
We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective.
Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.
Alternative splicing and transcriptional initiation are key mechanisms, which generate diversity both at the mRNA and protein levels. Recently, several independent research efforts revealed that more than 90% of human genes are alternatively spliced [1, 2], and about 50% of both human and mouse genes have multiple alternative promoters . Additionally, a genome-wide screening of alternative splicing and transcriptional initiation estimated that a significant number of genes are differentially spliced within 5' and 3' untranslated regions (UTRs) . Moreover, another genome-wide analysis identified 324 out of 17897 genes that display associations between flanking single nucleotide polymorphisms (SNPs) and gene expression/alternative transcription, demonstrating the regulatory effects of genetic variation in human populations . These non-bias/genome-wide analyses highlight the importance of alternative splicing/promoter usage as general mechanisms of regulation control in mammalian cells.
UTRs are considered to influence gene expression by modulating mRNA stability and/or translational efficiency. Consequently, UTR heterogeneity for a specific gene is likely to have a differential impact on protein expression . Analysis of variable 5' UTRs in the TGF-beta, BRCA1 and MDM2 genes, have indicated that the shorter UTR variants are translated more efficiently .
The Hedgehog (HH) signaling pathway plays a central role in embryonic development and adult tissue homeostasis . Abnormal activation of the pathway has been associated with various cancers in skin, brain, lung, digestive tract, prostate and pancreas [9–12]. The mechanistic details of the HH signaling pathway, which is generally thought to be well conserved in evolution, have mostly emerged from studies in Drosophila. In the absence of HH ligands, the PTCH receptor inhibits the activity of the 7-pass transmembrane protein Smoothened (SMO), which acts as a positive regulator of the pathway. Interaction of HH ligands with PTCH relieves the inhibitory control of PTCH on SMO, allowing the GLI transcription factor to dissociate from the negative regulator Suppressor of Fused (SUFU) and translocate into the nucleus, activating target genes. In mammals there are three paralogues of HH proteins, Sonic HH, Indian HH and Desert HH, two PTCH receptors, PTCH1 and PTCH2, and three GLI transcription factors, GLI1, GLI2 and GLI3. Additionally, splice variants of several HH signaling components have been identified , in line with an earlier report, which, by the use of genome-wide RNAi, highlighted the importance of alternative splicing in this pathway .
GLI1 was originally identified as a highly expressed gene in human glioma  and acts as a downstream effector of the HH signaling cascade, mediating the transcriptional response . GLI1 is also a target gene of the HH pathway, resulting in a positive feedback loop. Moreover, overexpression of GLI1 in transgenic mice leads to the induction of basal cell carcinomas and trichoepitheliomas . Alternative splicing in the GLI1 5' UTR regions of human and mouse has earlier been reported by Wang and Rothnagel . Recently, we and others have demonstrated the occurrence of functional, differential splicing events in the coding regions of human GLI1 [19, 20].
In this study, novel mouse Gli1 mRNA variants, whose transcriptional initiation is further upstream of the reported exon 1 sequence, were identified. Additionally, we obtained evidence that genetic variation is a key determining factor for the alternative splicing events in the Gli1 5' UTRs, which have functional implications on translational efficiency.
Identification of novel transcription start sites in the mouse Gli1 gene
Primer sequences for RACE and nested PCR analysis
Forward primers in exon 1
Reverse primers in exon 4
Expression profiles of Gli1 variants in embryos, cell lines, and medulloblastoma tumors
Primer sequences for real-time RT-PCR
5' - CATAAGCCCGGCACCCCCTCTCTA
5' - ACCCGCGAGAAGCGCAAACTTTTT
5' - ACGAGGGAAGTGAGCGGGAAGAGC
5' - ACCCGCGAGAAGCGCAAACTTTTT
5' - TTGTCCGCGCCTCTCCCACATACTA
5' - GGGCAGAAGCAGCCGTTCAGTCTT
5' - TTGTCCGCGCCTCTCCCACATACTA
5' - CCCATAGGGTCTCGGGGTCTCAAAC
Initially, we confirmed Gli1 variant expression in the C57BL/6 mouse embryos at 8.5 and 9.5 d.p.c. by real-time RT-PCR (Figure 2B). Similarly to the nested PCR analysis shown in figure 1B, Gli1 variants skipping exon 1B (ΔE1B) were more abundant to the ones including exon 1B (E1B) in both embryonic stages. Furthermore, the C ycle t hreshold (Ct) values of Gli1 variants that were transcribed from TSS-L and TSS-M (L+M) were nearly equal to the Ct values of either the ΔE1B or the exon 11 to 12 (E11-12) transcripts. This finding highlighted that the majority of the mouse Gli1 mRNAs initiate at upstream TSSs in the embryo, in agreement with the previous observations of the NIH3T3 cell line (Figure 1B). Additionally, to elucidate the in vivo distribution of these Gli1 transcripts in developing embryonic compartments, we analyzed 9.5 d.p.c. mouse embryos by whole mount in situ hybridization (See additional file 1). All probes used showed comparable distribution patterns of the variants, as also did a Gli1 3' end control riboprobe. These data suggested that the expression of all the alternative Gli1 mRNAs was likely to be controlled by similar developmental mechanisms.
Overactivated HH signaling and Gli1 overexpression play a central role in medulloblastoma tumorigenesis . This prompted us to investigate the expression profile of the Gli1 variants in medulloblastomas from the tumor-prone Sufu+/-Trp53-/- mice maintained on a C57BL/6 genetic background (Heby-Henricson, K, Bergström, Å, Rozell, B, Toftgård, R, Teglund, S, unpublished) by using real-time RT-PCR (Figure 3C). As anticipated, the expression of all Gli1 variants was remarkably upregulated in the tumor samples compared to normal cerebellum, but the relative expression pattern of the variants was not apparently influenced by tumorigenesis. Additionally, the ΔE1B variant was found to be expressed at higher levels than E1B, as in wtMEFs and Sufu-/- MEFs. Similar upregulation patterns of the Gli1 transcripts were also observed in medulloblastomas from Ptch1+/- mice maintained on a C57BL/6 genetic background (See additional file 2).
These results are therefore suggesting that neither HH signaling activation nor tumorigenesis preferentially affect the regulation of expression of the individual Gli1 variants. On the other hand there is a strong influence of the mouse strain/genotype. C57BL/6 mice predominantly express the ΔE1B variant, BALB/c mice the E1B variant, while mice with mixed genetic background have comparable expression of both. In line with these observations, a mouse cDNA panel from BALB/c mice consistently showed increased expression of the E1B relative to the ΔE1B variant (data not shown).
Identification of SNP and SINE polymorphisms involved in mouse Gli1 exon 1B inclusion/skipping
Functional analysis of the Gli1 5' untranslated regions
Finally, we investigated whether this differential capacity of the 5' UTRs is due to modulations of the mRNA stability or the translational efficiency. To achieve this, the endogenous Gli1 levels in wtMEFs were enriched by SAG, followed by inhibition of the process of transcription with actinomycin D treatment and subsequent mRNA measurements. The results revealed no significant differences in mRNA stability among the Gli1 variants (See additional file 4). Thus, it may be suggested that the alternative 5' UTRs are likely to regulate Gli1 protein levels via translational mechanisms.
Initially, 5' RACE and RT-PCR analysis clearly showed 5' variations in the Gli1 transcripts (Figure 1). However, the previously reported exon 2 - exon 3 skipping in human normal tissues/tumor cell lines , exon 3 - partial exon 4 skipping in human cancer cells and especially glioblastomas  and exon 1A - exon 1B skipping in mouse  could not be detected. Moreover, the 5' RACE analysis revealed the presence of some minor transcripts, apparently transcribed from alternative TSSs (data not shown, DDBJ: AB232673 - AB232676). As their expression levels were quite low, we chose to focus on the relatively abundant Gli1 variants in order to analyze the impact of alternative transcripts on HH signaling.
We demonstrated that exon 1B inclusion/skipping is intimately related with SNPs in the donor site of intron 1B (Figures 1, 2, 3, 4 and 5). The GT allele contains a canonical 5' splice site sequence, allowing efficient exon 1B definition and consequently enhances the inclusion of exon 1B, as observed in NIH3T3 cells. On the other hand, the GC allele has a weaker 5' splice site sequence, resulting in a less efficient exon 1B definition and therefore promotes the skipping of exon 1B, as observed in wtMEFs and Sufu-/- MEFs. Moreover, Ptch1-/- MEFs, which are heterozygous and contain both the GT and GC alleles, expressed comparable levels of the exon 1B-included and -skipped variants.
Since the relative expression pattern of the Gli1 variants was not affected by either HH signaling activity (Figure 3A and 3B) or tumorigenesis (Figure 3C)/embryogenesis (Figure 2B), the identified polymorphisms are primary determinants in controlling the splicing regulation of the Gli1 5' UTRs. SNPs/mutations affecting splicing have been reported for consensus splice sites, but were also identified at significant distances from splice junctions [28, 29]. Our own analysis of the 284 reported mutations of PTCH1 revealed the presence of 20 intronic splice changes . These findings highlight the importance of SNPs/mutations in altering splicing patterns, as these may occur not only at canonical splice sites but also at exonic/intronic splicing enhancers/silencers.
Additionally, evolutionary comparisons indicated that GC is likely to represent the ancestral intronic dinucleotide at the 5' splice site of intron 1B, since it is conserved in rat and other species, with the GT substitution and the SINE B2 insertion occurring at later stages (See additional file 5 and Figure 5). Interestingly, in the primate lineage the exon 1B region is characterized by an Alu insertion. These facts suggest that this genomic segment might be genetically unstable and a hotspot for transposon insertion.
5' UTRs are known to regulate protein expression via modulation of mRNA stability and/or translational efficiency . We analyzed the alternative Gli1 5' UTRs in various cell lines and found that 5' end shortening as well as skipping of exon 1B increased their capacity for heterologous protein expression (Figure 6). These observations are in line with previous claims purporting that shorter 5' UTRs of Gli1 are more capable of efficient translation , and support the notion that alternative events in 5' UTRs of mammalian genes are likely to contribute to the regulation of translation . Moreover, mRNA stability assays of the alternative Gli1 transcripts revealed that variations in the 5' UTRs did not affect the pattern of RNA degradation, and consequently, these untranslated sequences regulate Gli1 protein levels by modulating translational efficiency. Interestingly and in line with these observations, Hedgehog signaling-dependent mouse models for medulloblastoma development are apparently influenced by the Gli1 genotype. Deletion of one Ptch1 allele in C57BL/6 mice, which are homozygous for the GC allele, results in a higher incidence of medulloblastomas compared to mice with a mixed genetic background .
Secondary structure prediction of the Gli1 5' UTRs by Mfold highlighted the presence of a long stem-loop structure in L and M that is retained in MΔ1B but not in LΔ1B and may have a role in the differential translatabilities of MΔ1B versus LΔ1B (See additional file 3). However, free energy, ΔG, calculations were not fully in line with the capacity for translation. In addition, we examined whether two other parameters, G-quadruplex (G4) DNA/RNA structures and upstream ORF (upORFs), might influence the translational efficiency (See additional file 6). Nucleotide region 251-294 has a G4 motif sequence G3-N1-7-G3-N1-7-G3-N1-7-G3, and nucleotide region 22-55 has a similar sequence with a potential G4 structure. Moreover, the genome wide G4 DNA database QuadBase  predicted an antisense G4 motif at nucleotide region 322-360. G4-structures are formed not only on DNA but also on RNA , and a G4 motif on the NRAS mRNA was reported to suppress translation . Additionally, the exon 1B-included variants, L, M and S, have an upORF in the same frame as the Gli1 ORF (fully upstream, 46 encoded amino acids), while the exon 1B-skipped variants, LΔ1B, MΔ1B and SΔ1B, have an upORF (38 encoded amino acids) that overlaps with the Gli1 ORF. Although no significant differences between fully upstream and overlapping upORF could be identified in a recent report, long cap-to-upORF distances were found to increase translational inhibition . Wang and Rothnagel have used a 5' UTR construct (alfa-UTR), which is almost equivalent to the S construct in this report, mutated at four ATG codons and apparently eliminating 46 amino acids upORF that we have identified, and observed increased reporter activity, in line with the above predictions . Thus, the combinations of upORFs with G4 structures in the Gli1 5' UTRs are likely to have a role as mediators of the observed patterns of translation.
Our findings highlight the complex posttranscriptional regulation of the mouse Gli1 oncogene. mRNA variants with alternative 5' UTRs were identified, mechanisms that control their expression levels were dissected, and the differential impact of the 5' UTRs on protein synthesis was determined. Moreover, the demonstrated strain differences in regulatory controls of this oncogene suggest that these may have a role in modulating tumor susceptibility in mouse models.
RACE and PCR
5' RACE was performed by using the GeneRacer kit (Life Technologies, CA, USA), with mouse Gli1 exon 4 reverse primers (MWG-Biotech, Ebergsberg, Germany) (Table 1). The RACE products were analyzed on a 4% NuSieve 3:1 agarose gel (FMC BioProducts, ME, USA) and verified by PCR direct sequencing or sequencing of TA-clones in the pGEM-T vector (Promega, WI, USA). Pairs of initial and nested primers were also designed within mouse Gli1 exon 1, as shown in Table 1, and used in combination with the RACE exon 4 primers. The nested PCR analysis and sequence-verification were carried out as described in previous reports [37, 38].
The murine fibroblast cell lines NIH3T3, Ptch1-/- MEFs , wtMEFs, Sufu-/- MEFs and C3H/10T1/2 were cultured as described before [37, 40, 41]. Cells were treated with the S mo ag onist SAG at a concentration of 100 nM, with the medium changed to low serum (0.5% FBS or 1% FBS for wtMEFs), and allowed to grow for an additional 2 days.
The use of animals was approved by the Stockholm South Animal Ethics Committee. The mice were kept at the animal facility of the Karolinska University Hospital, according to local and national regulations. The Sufu+/-Trp53-/- mice were generated by intercrossing Sufu+/-  and Trp53+/- mice . The Ptch1+/- mouse strain has been described previously . Both the Sufu+/-Trp53-/- and the Ptch1+/- strains were maintained on a C57BL/6 genetic background.
Isolation of cerebellum cells
Normal cerebella and medulloblastoma tumors from Sufu+/-Trp53-/- mice were digested with papain, triturated to obtain single-cell suspensions and then centrifuged through a 35%-65% Percoll gradient. Cells from the 35%-65% interface were suspended in Neurobasal medium (Life Technologies, CA, USA). Isolated granule cells were counted and checked with a microscope.
RNA isolation, and real-time RT-PCR
Total RNA was isolated from cells, tissues and mouse embryos, using the RNeasy kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. Real-time RT-PCR was performed as described before . Dissociation curves were generated after each PCR run to ensure that a single, specific product was amplified. The results were analyzed with the comparative C ycle t hreshold (Ct) method. For normalization, we used the expression level of Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and/or Acidic ribosomal protein (Arp). The PCR primers are shown in Table 2.
Analysis of polymorphic variants
Primer sequences for polymorphism analysis
Functional analysis of 5' UTRs
Primer sequences for generation of the 5' UTR constructs
Two hundred ng of each of the 5' UTR constructs were transfected into NIH3T3 or wtMEF cells, with or without SAG treatment initiated 24 hours after transfection, and Ptch1-/- or Sufu-/- MEFs using the FuGENE6 (Roche Diagnostics, Basel, Switzerland) transfection reagent. The activities of Renilla and Firefly luciferases were determined by using the dual-luciferase reporter assay system (Promega) with a FB12 Luminometer (Berthold Detection System, Pforzheim, Germany) or an Infinite M200 (Tecan, Männedorf, Switzerland) according to the manufacturer's recommendations. The experiments and individual measurements were performed at least twice.
Glioma associated oncogene 1
Suppressor of Fused
acid ribosomal protein
days post coitum
long 5' UTR transcript
medium 5' UTR transcript
short 5' UTR transcript
L transcript with skipped exon 1B
M transcript with skipped exon 1B
S transcript with skipped exon 1B
single nucleotide polymorphism
We thank J. Svärd for help with the Ptch+/- medulloblastoma samples. Professor Rune Toftgård is acknowledged for generous support. We are grateful to V. Jaks, M. Kasper, I. Sur, C. Finta, R. Saraswathi and Å. Bergström for helpful discussions and technical support. This study was supported by the Swedish Cancer Fund, the Swedish Research Council, the Swedish Childhood Cancer Foundation and the Magnus Bergvalls Foundation. RP was partially supported by Karolinska Institutet' s funding for postgraduate students. TS was supported by a Marie Curie International Incoming Fellowship.
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