Abstract
The rat erbA alpha locus encodes two overlapping mRNAs, alpha 1 and alpha 2, which are identical except for their most 3' exons. alpha 1 mRNA encodes a thyroid hormone receptor, while alpha 2 encodes an altered ligand binding domain of unknown function. Previous studies have shown that the ratio of alpha 1 to alpha 2 is highest in cells expressing a high level of a third RNA, Rev-ErbA alpha mRNA, which is transcribed in the opposite direction and is complementary to alpha 2 but not alpha 1 mRNA. It was hypothesized that base pairing with Rev-ErbA alpha blocks splicing of alpha 2 mRNA, thereby favoring formation of the non-overlapping alpha 1. To test this model, a system was developed in which alpha 2 pre-mRNAs were accurately spliced in vitro. Splicing was inhibited by the addition of a 5-fold excess of antisense RNAs containing the 3' end of Rev-ErbA alpha mRNA. Both an antisense RNA extending across the 3' splice site and a shorter RNA complementary only to exon sequences efficiently blocked splicing. However, splicing was only inhibited by complementary RNAs. These observations are consistent with a mechanism in which base pairing with a complementary RNA regulates alternative processing of alpha 1 and alpha 2 mRNAs.
Highlights
Previous studies have shown that the ratio of a1 to a2 is highest in cells expressing a high level of a third RNA, Rev-ErbAa mRNA, which is transcribed in the opposite direction and is complementary to a2 but not a1 mRNA
It was hypothesized that base pairing with Rev-ErbAa blocks splicing of a2 mRNA, thereby favoring formation of binding or by forming inactive heterodimers [2, 20,21,22]
ErbAa, which is transcribed in the opposite direction from a1 and a2 [14,23]
Summary
Plasmids-The construction of plasmids pHB500 and pHBS has been described previously[27]. pHB-n2Lwas constructed by inserting the 775-nt HincIIIEcoRI fragment (A in Fig. 1A) which includes the a2-specific 3’ splice site from the rat erbAn locus [16, 23] into the same sites of pHBS, downstream of the @-globininsert. pHB-n2S was constructed by deleting the 473-nt HincIIISspI fragment from. PHB-n2Lwas constructed by inserting the 775-nt HincIIIEcoRI fragment (A in Fig. 1A) which includes the a2-specific 3’ splice site from the rat erbAn locus [16, 23] into the same sites of pHBS, downstream of the @-globininsert. PHB-n2S was constructed by deleting the 473-nt HincIIISspI fragment from. Pennsylvania Diabetes and Endocrine Research Center (to M.A. L.) areidenticaltothoseintheraterbAn gene. H. constructed by replacing the globin insert of pHB-n2L with the 510-. The abbreviations used are: T3, thyroid hormone;TR(s),thyroid short region of pGEM4 between the XbaI site of the insert and the hormone receptor(s); nt, nucleotide(s); pre-mRNA, mRNA precursoSr.phI siteof the vector. The abbreviations used are: T3, thyroid hormone;TR(s),thyroid short region of pGEM4 between the XbaI site of the insert and the hormone receptor(s); nt, nucleotide(s); pre-mRNA, mRNA precursoSr.phI siteof the vector. pn2-S was constructed by replacing fragment
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