The Drosophila U1 and U6 Gene Proximal Sequence Elements Act as Important Determinants of the RNA Polymerase Specificity of Small Nuclear RNA Gene Promoters in Vitroand in Vivo

Kathleen J Mcnamara-Schroeder,Roger F Hennessey,Gale A Harding,Richard C Jensen,William E Stumph

doi:10.1074/jbc.m101273200

Kathleen J Mcnamara-Schroeder, Roger F Hennessey + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m101273200

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Abstract

Transcription of genes coding for metazoan spliceosomal snRNAs by RNA polymerase II (U1, U2, U4, U5) or RNA polymerase III (U6) is dependent upon a unique, positionally conserved regulatory element referred to as the proximal sequence element (PSE). Previous studies in the organism Drosophila melanogaster indicated that as few as three nucleotide differences in the sequences of the U1 and U6 PSEs can play a decisive role in recruiting the different RNA polymerases to transcribe the U1 and U6 snRNA genes in vitro. Those studies utilized constructs that contained only the minimal promoter elements of the U1 and U6 genes in an artificial context. To overcome the limitations of those earlier studies, we have now performed experiments that demonstrate that the Drosophila U1 and U6 PSEs have functionally distinct properties even in the environment of the natural U1 and U6 gene 5'-flanking DNAs. Moreover, assays in cells and in transgenic flies indicate that expression of genes from promoters that contain the "incorrect" PSE is suppressed in vivo. The Drosophila U6 PSE is incapable of recruiting RNA polymerase II to initiate transcription from the U1 promoter region, and the U1 PSE is unable to recruit RNA polymerase III to transcribe the U6 gene.

Highlights

Genes coding for most of the small nuclear RNAs1 are transcribed by RNA polymerase II, but U6 genes are transcribed by RNA polymerase III
Exchanging the U1 and U6 proximal sequence element A (PSEA) Indicates That They Are Not Functionally Equivalent in Vitro—Previous transcription studies that examined the role of cis-acting elements in determining RNA polymerase specificity utilized synthetic DNA templates that contained only the essential Drosophila U1 or U6 promoter elements and lacked most of the remaining sequences present in the wild type U1 or U6 gene 5Ј-flanking DNA [20]. Those studies indicated that the U1 and U6 PSEAs could act as the major determinants of RNA polymerase specificity in vitro
Previous in vitro studies suggested that the RNA polymerase specificity of Drosophila snRNA genes was determined primarily by a few nucleotide differences within the 21-base pair PSEA sequence

Summary

EXPERIMENTAL PROCEDURES

In Vitro Transcription of U1 and U6 Gene Promoter Templates—The templates that contained the wild type U1 and U6 promoter sequences have been described previously [7, 21]. Expression of U6 Maxigene Constructs by Transfection of Drosophila Tissue Culture Cells—The plasmid pU6-maxi was a gift from Deborah Johnson (Departments of Molecular Pharmacology and Biochemistry, University of Southern California) This plasmid contained an insertion of 25 base pairs at position 66 in the U6 RNA coding region. NeoR Gene Expression Driven by the U1 Promoter in Transient Expression Assays—The neoR gene was obtained from the pP{hsneo} Pelement vector [23] ( known as pUChsneo [24]) by polymerase chain reaction with primers that flanked the neoR gene and contributed an upstream BclI site and a downstream BamHI site This fragment was cloned into the BamHI site of pBluescriptSK(ϩ) such that the SpeI site of the vector was upstream of the neoR gene, producing the construct pSK/neo. The constructs pSK/U1(wild type)/neo and pSK/U1(U6PSEA)/neo described above were each di-

RNA Polymerase Selection by the PSE at snRNA Gene Promoters

RESULTS

DISCUSSION

Fly line