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Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene.
RNA. 2004 Aug; 10(8):1243-50.RNA

Abstract

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.

Authors+Show Affiliations

Department of Biochemistry, Virginia Commonwealth University, 40l College Street, Richmond 23298, USA. jroesser@hsc.vcu.edu

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15272120

Citation

Roesser, James R.. "Both U2 snRNA and U12 snRNA Are Required for Accurate Splicing of Exon 5 of the Rat calcitonin/CGRP Gene." RNA (New York, N.Y.), vol. 10, no. 8, 2004, pp. 1243-50.
Roesser JR. Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene. RNA. 2004;10(8):1243-50.
Roesser, J. R. (2004). Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene. RNA (New York, N.Y.), 10(8), 1243-50.
Roesser JR. Both U2 snRNA and U12 snRNA Are Required for Accurate Splicing of Exon 5 of the Rat calcitonin/CGRP Gene. RNA. 2004;10(8):1243-50. PubMed PMID: 15272120.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene. A1 - Roesser,James R, PY - 2004/7/24/pubmed PY - 2004/8/28/medline PY - 2004/7/24/entrez SP - 1243 EP - 50 JF - RNA (New York, N.Y.) JO - RNA VL - 10 IS - 8 N2 - 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. SN - 1355-8382 UR - https://www.unboundmedicine.com/medline/citation/15272120/Both_U2_snRNA_and_U12_snRNA_are_required_for_accurate_splicing_of_exon_5_of_the_rat_calcitonin/CGRP_gene_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&amp;pmid=15272120 DB - PRIME DP - Unbound Medicine ER -