Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.
ABSTRACT The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.
- SourceAvailable from: Graham J G Upton[Show abstract] [Hide abstract]
ABSTRACT: We are developing a computational pipeline to use surveys of Affymetrix GeneChips as a discovery tool for unravelling some of the biology associated with post-transcriptional processing of RNA. This work involves the integration of a number of bioinformatics resources, from comparing annotations to processing images to determining the structure of transcripts. The rapidly growing datasets of GeneChips available to the community puts us in a strong position to discover novel biology about post-transcriptional processing, and should enable us to determine the mechanisms by which some groups of genes make co-ordinated changes in their production of isoforms.Biochemical Society Transactions 07/2008; 36(Pt 3):511-3. · 2.59 Impact Factor
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ABSTRACT: Affymetrix GeneChip technology enables the parallel observations of tens of thousands of genes. It is important that the probe set annotations are reliable so that biological inferences can be made about genes which undergo differential expression. Probe sets representing the same gene might be expected to show similar fold changes/z-scores, however this is in fact not the case. We have made a case study of the mouse Surf4, chosen because it is a gene that was reported to be represented by the same eight probe sets on the MOE430A array by both Affymetrix and Bioconductor in early 2004. Only five of the probe sets actually detect Surf4 transcripts. Two of the probe sets detect splice variants of Surf2. We have also studied the expression changes of the eight probe sets in a public-domain microarray experiment. The transcripts for Surf4 are correlated in time, and similarly the transcripts for Surf2 are also correlated in time. However, the transcripts for Surf4 and Surf2 are not correlated. This proof of principle shows that observations of expression can be used to confirm, or otherwise, annotation discrepancies. We have also investigated groups of probe sets on the RAE230A array that are assigned to the same LocusID, but which show large variances in differential expression in any one of three different experiments on rat. The probe set groups with high variances are found to represent cases of alternative splicing, use of alternative poly(A) signals, or incorrect annotations. Our results indicate that some probe sets should not be considered as unique measures of transcription, because the individual probes map to more than one transcript dependent upon the biological condition. Our results highlight the need for care when assessing whether groups of probe sets all measure the same transcript.BMC Bioinformatics 02/2007; 8:13. · 3.02 Impact Factor
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ABSTRACT: Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.Molecular and Cellular Biology 03/2007; 27(3):830-41. · 5.37 Impact Factor