A cloned human ribosomal protein gene functions in rodent cells.

Abstract

Cloned fragments of human ribosomal protein S14 DNA (RPS14) were transfected into cultured Chinese hamster (CHO) cells. Transient expression assays indicated that DNA with as little as 31 base pairs of upstream flanking sequence was transcribed into a polyadenylated, 650-base mRNA that is largely bound to the polyribosomes. In these respects the exogenous human S14 message appeared to function normally in CHO cells. Interestingly, transcription of human RPS14 did not require the TATA sequence located 26 base pairs upstream of exon 1. Stably transformed clones were selected from cultures of emetine-resistant CHO cells (Emr-2) after transfection with pSV2Neo-human RPS14 constructs. Human RPS14 complemented the mutationally based drug resistance of the Chinese hamster cells, demonstrating that the cloned human ribosomal protein gene is functional in rodent cells. Analysis of transformed cells with different amounts of integrated RPS14 indicated that human S14 mRNA levels are not tightly regulated by CHO cells. In contrast, the steady-state S14 level fluctuated only slightly, if at all, in transformed clones whose S14 message contents differed by more than 30-fold. These data support the conclusion that expression of human RPS14 is regulated, at least partially, posttranscriptionally.Ribosomal components are encoded by a complex family of genes in eucaryotic and procaryotic organisms. Because approximately equal amounts of ribosomal proteins (rproteins) and rRNAs are synthesized under various growth conditions (11,13,19,21,28,30,36,40,45), ribosomal genes appear to be controlled by stringent biochemical and genetic mechanisms. Expression of r-protein genes is governed by a combination of transcriptional, translational, and posttranslational mechanisms in bacteria and lower eucaryotes (12,15,20,29,33,35,39,43,44,(47)(48)(49). Comparable information about eucaryotic, especially mammalian, r-protein genes is sparse owing to a lack of high-resolution genetic systems.Only one of the mammalian r-protein genes so far isolated is amenable to genetic analysis in tissue culture. Recessive mutations (EmtB) affecting the CHO cell r-protein S14 gene (RPS14) result in resistance to emetine, an alkaloid inhibitor of protein biosynthesis (2,3,16,17,24). EmtB S14 protein is slightly more acidic than wild-type S14 (2), and ribosomes containing mutant S14 are relatively unstable (23, 45). RPS14 genomic and cDNA sequences have been cloned from Chinese hamster and human cells, and their complete nucleic acid sequences are known (32,37,38). Human RPS14 consists of five exons and four introns spanning 6 kilobase pairs (kbp) of chromosome Sq DNA (37). It contains a TATA box 28 bp upstream of exon 1. In this regard RPS14 differs from the few other well-characterized mammalian (mouse) r-protein genes, which all lack TATA sequences (9,42,46 protein. Our data demonstrated that the cloned DNAs encode a complete and functional r-protein gene. Disrupting the TATA sequence immediately 5' of exon 1 (37) did not preclude transcription of S14 mRNA, w...

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

“…Thus, despite experiments that demonstrate constitutive expression of mammalian r-protein genes in exponentially growing tissue culture cells (Rhoads and Roufa 1987), these observations indicate that under special physiological and tissue culture circumstances, mammalian r-protein genes are regulated dynamically at the level of transcription.…”

“…4, To analyze cell-free transcription of human RPS14 intron 1 further, we used plasmid pGANB ( Fig. 1) as a runRegulation of human RPS14 transcription off transcription template, pGANB includes all of the sequences necessary for accurate transient expression of S14 m R N A in cultured rodent cells (Rhoads and Roufa 1987;Overman et al 1993) and, based on the data in Figure 2, also should encode c~-250 and oL-280. Both sense and antisense transcripts synthesized in vitro were assayed by nuclease protection using complementary RNA probes.…”

“…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.…”

“…We have shown previously that expression of human RPS14 minigenes in cultured rodent cells requires at least 32 bp of proximal upstream DNA, the gene's 55-bp noncoding first exon, and a short (<300 bp) segment of its first intron (Rhoads et al 1986;Rhoads and Roufa 1987). 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 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).…”