Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene.
Two classes of spliceosome are present in eukaryotic cells. Most introns in nuclear pre-mRNAs are removed by a spliceosome that requires U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs). A minor class of introns are removed by a spliceosome containing U11, U12, U5, U4atac, and U6 atac snRNPs. We describe experiments that demonstrate that splicing of exon 5 of the rat calcitonin/CGRP gene requires both U2 snRNA and U12 snRNA. In vitro, splicing to calcitonin/ CGRP exon 5 RNA was dependent on U2 snRNA, as preincubation of nuclear extract with an oligonucleotide complementary to U2 snRNA abolished exon 5 splicing. Addition of an oligonucleotide complementary to U12 snRNA increased splicing at a cryptic splice site in exon 5 from <5% to 50% of total spliced RNA. Point mutations in a candidate U12 branch sequence in calcitonin/CGRP intron 4, predicted to decrease U12-pre-mRNA base-pairing, also significantly increased cryptic splicing in vitro. Calcitonin/CGRP genes containing base changes disrupting the U12 branch sequence expressed significantly decreased CGRP mRNA levels when expressed in cultured cells. Coexpression of U12 snRNAs containing base changes predicted to restore U12-pre-mRNA base pairing increased CGRP mRNA synthesis to the level of the wild-type gene. These observations indicate that accurate, efficient splicing of calcitonin/CGRP exon 5 is dependent upon both U2 and U12 snRNAs.
Department of Biochemistry, Virginia Commonwealth University, 40l College Street, Richmond 23298, USA. jroesser@hsc.vcu.edu
James R
JR
Roesser
D000818:Animals; D001483:Base Sequence; D002116:Calcitonin / Q000096:biosynthesis / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D008969:Molecular Sequence Data; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D051381:Rats
D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C096986:U12 small nuclear RNA; C405484:U2 small nuclear RNA; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide