Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element.

Abstract

The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells.This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selective translational control is not confined to rp mRNAs, as mRNAs encoding the naturally occurring ubiquitin-rp fusion protein and elongation factor la, which contain a 5' TOP, also conform this mode of regulation; (iii) rat rpP2 mRNA contains only five pyrimidines in its 5' TOP, yet this mRNA is translationally controlled in the same fashion as other rp mRNAs with a 5' TOP of eight or more pyrimidines; (iv) full manifestation of this mode of regulation seems to require both the 5' TOP and sequences immediately downstream; and (v) an intact translational regulatory element from rpL32 mRNA fails to exert its regulatory properties even when preceded by a single A residue.The translation of vertebrate ribosomal protein (rp) mRNAs is largely regulated in a growth-dependent manner, as illustrated by their selective release from polysomes in growtharrested cells. This has been demonstrated during transition of various cells types between growing and nongrowing states in response to a wide variety of physiological stimuli, including dexamethasone-treated mouse lymphosarcoma cells (45,48), serum starvation of mouse fibroblasts (24,38) or Xenopus kidney cells (41), and differentiation of mouse myoblasts (1) and concanavalin A-treated bovine T cells (37). Similar specific translational fluctuations have been observed for rp mRNAs during the transition from the rapidly growing state in the fetal liver to the quiescent state in the adult and upon resumption of the hepatocyte proliferation in the regenerating liver (2). The mammalian, avian, and amphibian rp mRNAs that have been rigorously analyzed thus far contain a 5'-terminal oligopyrimidine tract (5' TOP), which consists of a C residue at the cap site, followed by an uninterrupted sequence of up to 13 pyrimidines (3,18,54,69,71). In attempt to delimit the translational regulatory element (TLRE), it has been shown that the 35-nucleotide-long 5' untranslated region (UTR) of Xenopus rpS19 is sufficient to confer translational repression on a chimeric mRNA in a developmental stage-dependent manner (43). The involv...

The Translational Cis ‐Regulatory Element of Mammalian Ribosomal Protein mRNAs is Recognized by the Plant Translational Apparatus

“…To obtain growth hormone mRNAs with the desired 5' termini, growth hormone genes driven by mouse ribosomal protein promoters (wild type or modified) or rat p-actin promoter were initially constructed. These chimeric genes were stably transfected into NIH 3T3 cells and the transcription start site for each construct was identified by primer-extension analysis [5]. The poly(A)-rich RNA isolated from these cells was translated in wheat-germ extract and the synthesis of growth hormone, determined by RIA, was established to be linearly proportional to the amount of added poly(A)-rich RNA up to at least 12 pg/ml (data not shown).…”

“…NIH 3T3 mouse fibroblasts were grown, transfected and growth-arrested as previously described [5,111. The amount of human growth hormone secreted from cells transfected with growth hormone chimeric genes was measured by RIA with a commercial kit (Nichols Institute).…”

Lithium Can Relieve Translational Repression of TOP mRNAs Elicited by Various Blocks along the Cell Cycle in a Glycogen Synthase Kinase-3- and S6-Kinase-independent Manner

“…Translational activation of TOP mRNAs is apparent upon resumption of cell division when resting cells are induced to grow (increase in their mass) or when amino acid-starved cells are refed (4,5). It is noteworthy that human lymphoblastoid cell lines exhibit constitutive translational repression of TOP mRNAs, even when mitotically active (5)(6)(7).…”

Growth-dependent and PKC-mediated translational regulation of the upstream stimulating factor-2 (USF2) mRNA in hematopoietic cells

“…Cell division was arrested by treatment with 0.1 mM dexamethasone (Sigma) for 24 h as previously described (Avni et al, 1994). Mouse erythroleukemia (MEL) cells were also grown in suspension.…”

“…A 0.97 kb fragment bearing the ribosomal protein L-30 processed gene (Meyuhas et al, 1987), linking the 5' and 3' regions of the p3A fragment, was used as a control for loading an equivalent amount of total RNA. A 2.2 kb fragment of mouse b-actin (Avni et al, 1994) and a 1.9 kb fragment of USF1 (Meier et al, 1994) were also used as probes for this study. The DNA fragments were labeled with 32 P-dCTP (Amersham) using the random primed labeling technique (Feinberg and Vogelstein, 1983) up to a speci®c activity of 3610 8 c.p.m./mg.…”