Howard M. Fried

Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover.

The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.

Constitutive transcription of yeast ribosomal protein gene TCM1 is promoted by uncommon cis- and trans-acting elements.

The DNA sequence UAST (TCGTTTTGTACGTTTTTCA) was found to mediate transcription of yeast ribosomal protein gene TCM1. UAST was defined as a transcriptional activator on the basis of loss of transcription accompanying deletions of all or part of UAST, orientation-independent restoration of transcription promoted by a synthetic UAST oligomer inserted either into TCM1 or into the yeast CYC1 gene lacking its transcriptional activation region, and diminished transcription following nucleotide alterations in UAST. UAST bound in vitro to a protein denoted TAF (TCM1 activation factor); TAF was concluded to be a transcriptional activator protein because nucleotide alterations in UAST that diminished transcription in vivo also diminished TAF binding in vitro. The sequence of UAST bore no obvious resemblance to UASrpg, the principal cis-acting element common to most yeast ribosomal protein genes. Likewise, TAF was distinguished from the UASrpg-binding protein TUF, since (i) TAF and TUF were chromatographically separable, (ii) binding of either TAF or TUF to its corresponding UAS was unaffected by an excess of UASrpg or UAST DNA, respectively, and (iii) photochemical cross-linking experiments showed that TAF was a protein of 147 kilodaltons (kDa), while TUF was detected as an approximately 120-kDa polypeptide, consistent with its known size. Cross-linking experiments also revealed that both UAST and UASrpg bound a second heretofore unobserved 82-kDa protein; binding of this additional protein appeared to require binding of TAF or TUF. On the basis of the biochemical characterization of TAF and a lack of sequence similarity between UAST and UASrpg, we suggest that transcription of TCM1 is mediated by a cis-acting sequence and at least one trans-acting factor different from the elements which promote transcription of most other ribosomal protein genes. A second trans-acting factor may be shared by TCM1 and other ribosomal protein genes; this factor could mediate coordinate regulation of these genes.

Characterization of yeast strains with conditionally expressed variants of ribosomal protein genes tcm1 and cyh2.

We placed a regulatory sequence derived from the GAL10 locus of Saccharomyces cerevisiae at various distances from the start sites of transcription of two yeast ribosomal protein genes, tcm1 and cyh2. The hybrid ribosomal protein genes were transcribed at wild-type levels in the presence of galactose. In the absence of galactose, the hybrid genes were transcribed either at a reduced level or essentially not at all. Yeast cells which transcribe the ribosomal protein genes at a reduced rate continued to grow, suggesting that enhanced translation of the ribosomal protein mRNA may permit an adequate rate of synthesis of the corresponding protein. Consistent with this suggestion is the finding that preexisting mRNA decayed at a reduced rate when transcription was halted abruptly by removal of galactose. Yeast cells unable to transcribe tcm1 or cyh2 without galactose did not grow. These conditional lethal strains demonstrate that the ribosomal proteins encoded by tcm1 and cyh2 are essential; furthermore, these strains are potentially useful for isolating mutations in the tcm1 and cyh2 proteins affecting their transport, assembly, or function.

Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation.

When present in excess, the mRNAs for Saccharomyces cerevisiae ribosomal proteins L3 and L29 are translated less efficiently, so that synthesis of these proteins remains commensurate with that of other ribosomal proteins (N.J. Pearson, H.M. Fried, and J.R. Warner, Cell 29:347-355, 1982; J.R. Warner, G. Mitra, W.F. Schwindinger, M. Studeny, and H.M. Fried, Mol. Cell. Biol. 5:1512-1521, 1985). We used a yeast strain with a conditionally transcribed derivative of the L3 gene to deplete cells progressively of L3 mRNA. In this case translation of L3 mRNA did not become more efficient so that L3 was not maintained at a normal level. Even when there was an initial excess of L3 mRNA, interruption of its further transcription produced an immediate drop in L3 synthesis, suggesting that the translational efficiency of preexisting mRNA cannot be altered. Lack of L3 synthesis afforded an opportunity to examine coordinate accumulation of other ribosomal proteins. Without L3, apparent synthesis of several 60S subunit proteins diminished, and 60S subunits did not assemble. A similar phenomenon occurred when, in a second strain, synthesis of ribosomal protein L29 was prevented. Loss of 60S subunit assembly was accompanied by a destabilization of some 60S ribosomal protein mRNAs. These data suggest that synthesis of some S. cerevisiae ribosomal proteins may be regulated posttranscriptionally as a function of the extent to which they are assembled.

Additional layers of gene regulatory complexity from recently discovered microRNA mechanisms

In recent years microRNAs have become recognized as pervasive, versatile agents of gene regulation. Some widely embraced rules involving Watson-Crick hybridization of microRNAs with mRNAs have generated great interest as scientists envision potential RNA cargoes for gene therapy and other experimental systems. However, while researchers ardently seek simplifying principles, nature seems very uncooperative. This article reviews some small RNA mechanisms that potentially regulate genes and which are not covered by previous microRNAs characterizations. In addition, we report here results of fluorescence microscopy experiments to directly demonstrate nuclear import of small RNAs equal in length to typical mature microRNAs, implying that gene regulation at the locus of transcription might be possible.

Translational Regulation of Ribosomal Protein Gene Expression in Eukaryotes

The ribosome has attracted our attention not only because of its remarkable ability to polymerize amino acids under the guidance of ribonucleotide triplets, but also because of the relationship between synthesis and assembly of its constituents. In eukaryotes, the ribosome is assembled from four RNA molecules and between 70 and 80 proteins.1 These various components are distributed over two subunits, one comprising roughly one third and the other two thirds of the total ribosome mass. Accumulation of these components, which may represent as much as 85% of a cell’s RNA and 15% of its protein, is an extremely efficient process. How is it that all the macromolecules in such a diverse collection exhibit similar properties of synthesis or accumulation to fulfill the exacting demands of their association? It is becoming increasingly evident that in eukaryotes, posttranscriptional regulatory mechanisms play a role in bringing about and maintaining the appropriate accumulation of ribosomal proteins. Fittingly, regulation of the translation of ribosomal protein messenger RNAs (mRNAs) appears to be one such mechanism. This chapter constitutes a summary of our current knowledge of these mechanisms along with some thoughts for future investigation.

Nucleocytoplasmic Transport in Ribosome Biogenesis

The assembly of ribosomes in eukaryotic cells affords numerous opportunities to investigate mechanisms of nuclear-cytoplasmic transport. All stages of the assembly process involve both RNA and protein molecules moving into the nucleus, some being exported out, and still others being exported from the nucleus only to re-enter subsequently. What are the basic mechanisms that bring about the proper subcellular localization of ribosomal and non-ribosomal components involved in ribosome assembly? Are there aspects to the transport of ribosomal materials unique to these macromolecules and their multicomponent complexes or does nuclear-cytoplasmic transport of ribosomal components occur through the same pathways and mechanisms applicable to other cellular constituents? In addition, ribosome assembly takes place amid little if any pool of free ribosomal components; correspondingly, should a component either be produced in excess or prevented from assembly (usually through artificial conditions created by molecular biologists) that component is degraded very rapidly (Larson et al, 1991); thus, one might also ask whether there are mechanisms that coordinate the nuclear-cytoplasmic traffic of ribosomal components and accessory assembly factors.