Duplicated ribosomal protein paralogs promote alternative translation and drug resistance.

Ribosomes are often seen as monolithic machines produced from uniformly regulated genes. However, in yeast most ribosomal proteins come from duplicated genes. Here, we demonstrate that gene duplication may serve as a stress-adaptation mechanism modulating the global proteome through the differential expression of ribosomal protein paralogs. Our data indicate that the yeast paralog pair of the ribosomal protein L7/uL30 produces two differentially acetylated proteins. Under normal conditions most ribosomes incorporate the hypo-acetylated major form favoring the translation of genes with short open reading frames. Exposure to drugs, on the other hand, increases the production of ribosomes carrying the hyper-acetylated minor paralog that increases translation of long open reading frames. Many of these paralog-dependent genes encode cell wall proteins that could promote tolerance to drugs as their translation increases after exposure to drugs. Together our data suggest a mechanism of translation control that functions through a differential use of near-identical ribosomal protein isoforms.

Regulation of ribosomal protein genes: An ordered anarchy.

Ribosomal protein genes are among the most highly expressed genes in most cell types. Their products are generally essential for ribosome synthesis, which is the cornerstone for cell growth and proliferation. Many cellular resources are dedicated to producing ribosomal proteins and thus this process needs to be regulated in ways that carefully balance the supply of nascent ribosomal proteins with the demand for new ribosomes. Ribosomal protein genes have classically been viewed as a uniform interconnected regulon regulated in eukaryotic cells by target of rapamycin and protein kinase A pathway in response to changes in growth conditions and/or cellular status. However, recent literature depicts a more complex picture in which the amount of ribosomal proteins produced varies between genes in response to two overlapping regulatory circuits. The first includes the classical general ribosome-producing program and the second is a gene-specific feature responsible for fine-tuning the amount of ribosomal proteins produced from each individual ribosomal gene. Unlike the general pathway that is mainly controlled at the level of transcription and translation, this specific regulation of ribosomal protein genes is largely achieved through changes in pre-mRNA splicing efficiency and mRNA stability. By combining general and specific regulation, the cell can coordinate ribosome production, while allowing functional specialization and diversity. Here we review the many ways ribosomal protein genes are regulated, with special focus on the emerging role of posttranscriptional regulatory events in fine-tuning the expression of ribosomal protein genes and its role in controlling the potential variation in ribosome functions. This article is categorized under: Translation > Ribosome Biogenesis Translation > Ribosome Structure/Function Translation > Translation Regulation.

Differential expression of duplicated ribosomal protein genes modifies ribosome composition in response to stress.

In Saccharomyces cerevisiae, most ribosomal proteins are synthesized from duplicated genes, increasing the potential for ribosome heterogeneity. However, the contribution of these duplicated genes to ribosome production and the mechanism determining their relative expression remain unclear. Here we demonstrate that in most cases, one of the two gene copies generate the bulk of the active ribosomes under normal growth conditions, while the other copy is favored only under stress. To understand the origin of these differences in paralog expression and their contribution to ribosome heterogeneity we used RNA polymerase II ChIP-Seq, RNA-seq, polyribosome association and peptide-based mass-spectrometry to compare their transcription potential, splicing, mRNA abundance, translation potential, protein abundance and incorporation into ribosomes. In normal conditions a post-transcriptional expression hierarchy of the duplicated ribosomal protein genes is the product of the efficient splicing, high stability and efficient translation of the major paralog mRNA. Exposure of the cell to stress modifies the expression ratio of the paralogs by repressing the expression of the major paralog and thus increasing the number of ribosomes carrying the minor paralog. Together the data indicate that duplicated ribosomal protein genes underlie a modular network permitting the modification of ribosome composition in response to changing growth conditions.

Promoter-dependent nuclear RNA degradation ensures cell cycle-specific gene expression.

Cell cycle progression depends on phase-specific gene expression. Here we show that the nuclear RNA degradation machinery plays a lead role in promoting cell cycle-dependent gene expression by triggering promoter-dependent co-transcriptional RNA degradation. Single molecule quantification of RNA abundance in different phases of the cell cycle indicates that relative curtailment of gene expression in certain phases is attained even when transcription is not completely inhibited. When nuclear ribonucleases are deleted, transcription of the Saccharomyces cerevisiae G1-specific axial budding gene AXL2 is detected throughout the cell cycle and its phase-specific expression is lost. Promoter replacement abolished cell cycle-dependent RNA degradation and rendered the RNA insensitive to the deletion of nuclear ribonucleases. Together the data reveal a model of gene regulation whereby RNA abundance is controlled by promoter-dependent induction of RNA degradation.

Introns are mediators of cell response to starvation.

Introns are ubiquitous features of all eukaryotic cells. Introns need to be removed from nascent messenger RNA through the process of splicing to produce functional proteins. Here we show that the physical presence of introns in the genome promotes cell survival under starvation conditions. A systematic deletion set of all known introns in budding yeast genes indicates that, in most cases, cells with an intron deletion are impaired when nutrients are depleted. This effect of introns on growth is not linked to the expression of the host gene, and was reproduced even when translation of the host mRNA was blocked. Transcriptomic and genetic analyses indicate that introns promote resistance to starvation by enhancing the repression of ribosomal protein genes that are downstream of the nutrient-sensing TORC1 and PKA pathways. Our results reveal functions of introns that may help to explain their evolutionary preservation in genes, and uncover regulatory mechanisms of cell adaptations to starvation.

Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein.

Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress.

Transcriptome wide annotation of eukaryotic RNase III reactivity and degradation signals.

Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions.

Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency.

Small nucleolar RNAs (snoRNAs) are among the first discovered and most extensively studied group of small non-coding RNA. However, most studies focused on a small subset of snoRNAs that guide the modification of ribosomal RNA. In this study, we annotated the expression pattern of all box C/D snoRNAs in normal and cancer cell lines independent of their functions. The results indicate that C/D snoRNAs are expressed as two distinct forms differing in their ends with respect to boxes C and D and in their terminal stem length. Both forms are overexpressed in cancer cell lines but display a conserved end distribution. Surprisingly, the long forms are more dependent than the short forms on the expression of the core snoRNP protein NOP58, thought to be essential for C/D snoRNA production. In contrast, a subset of short forms are dependent on the splicing factor RBFOX2. Analysis of the potential secondary structure of both forms indicates that the k-turn motif required for binding of NOP58 is less stable in short forms which are thus less likely to mature into a canonical snoRNP. Taken together the data suggest that C/D snoRNAs are divided into at least two groups with distinct maturation and functional preferences.

RNA-dependent regulation of the cell wall stress response.

Stress response requires the precise modulation of gene expression in response to changes in growth conditions. This report demonstrates that selective nuclear mRNA degradation is required for both the cell wall stress response and the regulation of the cell wall integrity checkpoint. More specifically, the deletion of the yeast nuclear dsRNA-specific ribonuclease III (Rnt1p) increased the expression of the mRNAs associated with both the morphogenesis checkpoint and the cell wall integrity pathway, leading to an attenuation of the stress response. The over-expression of selected Rnt1p substrates, including the stress associated morphogenesis protein kinase Hsl1p, in wild-type cells mimicked the effect of RNT1 deletion on cell wall integrity, and their mRNAs were directly cleaved by the recombinant enzyme in vitro. The data supports a model for gene regulation in which nuclear mRNA degradation optimizes the cell response to stress and links it to the cell cycle.

Deletion of Rnt1p alters the proportion of open versus closed rRNA gene repeats in yeast.

In Saccharomyces cerevisiae, the double-stranded-RNA-specific RNase III (Rnt1p) is required for the processing of pre-rRNA and coprecipitates with transcriptionally active rRNA gene repeats. Here we show that Rnt1p physically interacts with RNA polymerase I (RNAPI) and its deletion decreases the transcription of the rRNA gene and increases the number of rRNA genes with an open chromatin structure. In contrast, depletion of ribosomal proteins or factors that impair RNAPI termination did not increase the number of open rRNA gene repeats, suggesting that changes in the ratio of open and closed rRNA gene chromatin is not due to a nonspecific response to ribosome depletion or impaired termination. The results demonstrate that defects in pre-rRNA processing can influence the chromatin structure of the rRNA gene arrays and reveal links among the rRNA gene chromatin, transcription, and processing.

Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division.

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.

A physical interaction between Gar1p and Rnt1pi is required for the nuclear import of H/ACA small nucleolar RNA-associated proteins.

During rRNA biogenesis, multiple RNA and protein substrates are modified and assembled through the coordinated activity of many factors. In Saccharomyces cerevisiae, the double-stranded RNA nuclease Rnt1p and the H/ACA snoRNA pseudouridylase complex participate in the transformation of the nascent pre-rRNA transcript into 35S pre-rRNA. Here we demonstrate the binding of a component of the H/ACA complex (Gar1p) to Rnt1p in vivo and in vitro in the absence of other factors. In vitro, Rnt1p binding to Gar1p is mutually exclusive of its RNA binding and cleavage activities. Mutations in Rnt1p that disrupt Gar1p binding do not inhibit RNA cleavage in vitro but slow RNA processing, prevent nucleolar localization of H/ACA snoRNA-associated proteins, and reduce pre-rRNA pseudouridylation in vivo. These results demonstrate colocalization of various components of the rRNA maturation complex and suggest a mechanism that links rRNA pseudouridylation and cleavage factors.