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An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element

Published online by Cambridge University Press:  25 September 2001

ASOKA K. AMARASINGHE
Affiliation:
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA
ROBIN MacDIARMID
Affiliation:
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA Present address: Department of Plant and Microbial Biology, University of California, Berkeley, 111 Koshland Hall, Berkeley, California 94720, USA.
MELISSA D. ADAMS
Affiliation:
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA Present address: Department of Biology, University of North Carolina–Chapel Hill, 303 Fordham Hall, Chapel Hill, North Carolina 27599, USA.
DONALD C. RIO
Affiliation:
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA
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Abstract

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5′ exon. This element contains two pseudo-5′ splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5′ splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5′ exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5′ splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

Type
Research Article
Information
RNA , Volume 7 , Issue 9 , September 2001 , pp. 1239 - 1253
Copyright
2001 RNA Society

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