Multiple regulatory elements ensure accurate transcription of a human ribosomal protein gene

Abstract

Previously we have shown that expression of a cloned human ribosomal protein gene, RPS14, depends upon regulatory sites located within the gene's proximal upstream DNA plus its first intron. In order to identify cis-active sequence motifs within the RPS14 promoter-enhancer complex, we transiently expressed a set of informative deletion clones in cultured Chinese hamster ovary cells. These experiments revealed three DNA sequence motifs that surround the S14 mRNA initiation site and are necessary for accurate transcription. Electrophoretic mobility shift, DNase I footprint, and methylation interference assays resolved two nuclear proteins, NF alpha-1 and NF beta-1, which bind specifically to these regulatory motifs. NF-alpha 1 recognizes a pair of 6-bp target motifs (5'-TTCCGG-3') that flank the 5' end of RPS14 exon I; and NF-beta 1 binds to a 10-bp target sequence (5'-CCGTGGGAAC-3') within the gene's first intron. Site-directed deletion mutations within the NF-alpha 1 and -beta 1 binding sites markedly inhibit S14 mRNA transcription.

Suppression of human ribosomal protein L23A expression during cell growth inhibition by interferon-β

“…However, transcriptional control of rp genes has been observed during mouse myoblast dierentiation, suggesting the presence of regulatory elements in the promoter regions of the rp genes in muscle cells that may be aected by dierentiation status (Agrawal and Bowman, 1987). Moreover, NFa1 (E2F) and NF-b1 recognition sequences have been found in diverse mammalian housekeeping genes including ribosomal proteins (Overman et al, 1993). These results support the hypothesis that multiple mechanisms may underlie the regulation of ribosomal proteins during cell growth arrest and dierentiation in dierent cell types.…”

Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14.

“…Although large deletion mutations surrounding the NF-~31-binding site (such as between the BamHI and SmaI sites illustrated in Fig. 1) abrogated S14 transcription completely, site-specific deletions within the ~31 protein-binding site reduced transcription only partially {Overman et al 1993). For this reason, we proposed that the RPS14 downstream regulatory array is likely to be composed of multiple, cis-active functional elements, only one of which (the 131-binding site) is detected by electrophoretic methods used conventionally to assess DNA-protein interactions.…”

“…Previously, we had reported that full-length S14 mRNA is the only transcript initiated from the sense strand of this human chromosome segment (Rhoads and Roufa 1987;Overman et al 1993). Therefore, our current experiments focused on a search for antisense RNAs that might derive from the RPS14 regulatory intron.…”

“…DNA footprinting and electrophoretic mobility shift assays (EMSAs) of RPS14 minigenes carrying chemically defined deletion, insertion, and base substitution mutations indicated that RPS14 carries cis-active DNA regulatory motifs located both up-and downstream of its mRNA initiation site {Overman et al 1993). The upstream regulatory elements consist of at least the pair of similar DNA sequences (5'-CCGGAAR-3') that cooperatively bind transcription factor E2F (see Fig.…”

“…The human RPS14 locus encodes ribosomal protein S14 and is especially well suited for these studies. It is the only mammalian r-protein locus in which a drug selection system facilitates somatic genetics (Boersma et al 1979;Madjar et al 1982Madjar et al , 1983Diaz et al 1990;Diaz and Roufa 1992;Tasheva and Roufa 1993) and for which both genomic and eDNA clones carrying numerous wild-type and mutant alleles are available (Rhoads et al 1986;Rhoads and Roufa 1987;Diaz et al 1990;Overman et al 1993).…”