title: RNA secondary structure: an important cis-element in rat calcitonin/CGRP pre-messenger RNA splicing.
abstract: The calcitonin/CGRP gene pre-mRNA is alternatively spliced in a tissue-specific manner resulting in the formation of calcitonin mRNA in thyroid C-cells and CGRP mRNA in neurons. Computer analysis of the RNA containing the 3' splice acceptor of the calcitonin-specific exon 4 predicts that this region has the potential to form a thermodynamically stable stem-loop. Data from CD spectroscopy and solution phase structure probing with single-strand specific and double-strand specific RNases indicates that RNA in this region is substantially double stranded. In vitro splicing of chimeric human beta-globin/calcitonin transcripts in HeLa nuclear extract was inhibited by base changes predicted to destabilize the stem, while compensatory base changes resulted in splicing at 50% of wild-type levels. Changing the residue opposite the AG dinucleotide adenosine in the stem from G to U, allowing the formation of an A-U basepair, abolished usage of this splice acceptor in vitro. These results indicate that a thermodynamically stable RNA stem-loop forms in vitro at the 3' splice acceptor of exon 4 of the calcitonin/CGRP gene transcript. This RNA secondary structure acts as a novel cis-element involved in proper splice site selection.
authors.affiliation: Department of Biochemistry, Virginia Commonwealth University, Richmond 23227, USA.
authors.forename: T P
authors.initials: TP
authors.lastname: Coleman
authors.forename: J R
authors.initials: JR
authors.lastname: Roesser
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002116:Calcitonin / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics*; D002942:Circular Dichroism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism
chemical_list: D012322:RNA Precursors; D012313:RNA; D002116:Calcitonin; D012260:Ribonucleases; D015740:Calcitonin Gene-Related Peptide