Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.
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.
Coleman
Timothy P
TP
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA 23298, USA.
Tran
Quincy
Q
Roesser
James R
JR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002116:Calcitonin / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005091:Exons; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D011506:Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012313:RNA; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide