The 3'-untranslated region directs ribosomal protein-encoding mRNAs to specific cytoplasmic regions

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. We have analyzed the subcellular distribution of mRNAs encoding human cytosolic and mitochondrial ribosomal proteins. Biochemical fractionation experiments showed that the transcripts for cytosolic ribosomal proteins associate preferentially with the cytoskeleton via actin microfilaments. Transfection in HeLa cells of a GFP reporter construct containing the cytosolic ribosomal protein L4 3'-UTR showed that the 3'-UTR is necessary for the association of the transcript to the cytoskeleton. Using confocal analysis we demonstrate that the chimeric transcript is specifically associated with the perinuclear cytoskeleton. We also show that mRNA for mitochondrial ribosomal protein S12 is asymmetrically distributed in the cytoplasm. In fact, this transcript was localized mainly in the proximity of mitochondria, and the localization was 3'-UTR-dependent. In summary, ribosomal protein mRNAs constitute a new class of localized transcripts that share a common localization mechanism

POSSIBLE ROLE OF CRY1 AND CRY2 IN ORAL CARCINOGENESIS

Aim. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. It has been shown recently that the occurrence, development, prognosis, and treatment of cancer are closely related to the abnormal expression of certain circadian-clock genes. CRY1 and CRY2 circadian-clock gene plays an important role in the regulation of many normal hysiological rhythms. This proteins act as light-independent inhibitors of CLOCK-BMAL1 components of the circadian clock. It has been revealed recently that abnormal expression of CRY1 and CRY2 correlate closely with the occurrence and development of many cancers. However, the expression and significance of this proteins in oral squamous cell carcinoma (OSCC) remains unknown. The aim of this study was to evaluate the expression levels of CRY1 and CRY2 in oral cancer. Materials and methods. CRY1 and CRY2 expression in cancerous and peritumoral tissues (when it was present) from 27 patients with OSCC was detected by immunohistochemistry techniques. Of all samples were received medical records (age, sex, grading, TNM, site of localization of the tumor). Immunohistochemistry was then performed on two sections for each of 27 sample mounted on poly-Llysine-coated glass slides to evaluate respectively the expression of CRY1 and CRY2.Results. In this study, out of the 27 cases, 11 were +/- positive in tumor area for CRY1 (most of which are well (differentiated), while out of 23 cases in which we evaluated the peritumoral tissue present in the section, 18 were positive. Also in the cases of positive tumor, almost always cytoplasmic, the CRY1 appears to be more strongly positive in dysplastic areas or even more in healthy epithelium, with a negative regulation in the areas most undifferentiated. As for the CRY2, out of the 27 cases analyzed, 17 were positive in the tumor area while about 23 cases in which we evaluated in peritumoral tissue present in the sections, 20 cases were positive. In tumor epithelium were found positivity also medium / high, present in tumors of different degree of differentiation, in some cases in other nuclear or cytoplasmic and nuclear/cytoplasmic, but when present the CRY2 is expressed, in most cases, in a manner similar or more intensely in peritumoral dysplastic epithelium. In the case of CRY2, there were no positivity in healthy epithelium (when present), but only in dysplastic epithelium. In addition, the positivity observed especially in peritumoral epithelium were present in states intermediate/surface. Conclusions. In conclusion, abnormal expression levels of CRY1 and CRY2 in OSCC tissue compared to healthy or dysplastic tissue may be related to the process of tumorigenesis. Further research focusing on these genes may, from the perspective of biological rhythms, provide novel ideas and methods for a better understanding of the occurrence and development of tumors, and for treatment of oral cancer

Using Ribosomal Protein Genes as Reference: A Tale of Caution

Background: Housekeeping genes are needed in every tissue as their expression is required for survival, integrity or duplication of every cell. Housekeeping genes commonly have been used as reference genes to normalize gene expression data, the underlying assumption being that they are expressed in every cell type at approximately the same level. Often, the terms "reference genes'' and "housekeeping genes'' are used interchangeably. In this paper, we would like to distinguish between these terms. Consensus is growing that housekeeping genes which have traditionally been used to normalize gene expression data are not good reference genes. Recently, ribosomal protein genes have been suggested as reference genes based on a meta-analysis of publicly available microarray data. Methodology/Principal Findings: We have applied several statistical tools on a dataset of 70 microarrays representing 22 different tissues, to assess and visualize expression stability of ribosomal protein genes. We confirmed the housekeeping status of these genes, but further estimated expression stability across tissues in order to assess their potential as reference genes. One- and two-way ANOVA revealed that all ribosomal protein genes have significant expression variation across tissues and exhibit tissue-dependent expression behavior as a group. Via multidimensional unfolding analysis, we visualized this tissue-dependency. In addition, we explored mechanisms that may cause tissue dependent effects of individual ribosomal protein genes. Conclusions/Significance: Here we provide statistical and biological evidence that ribosomal protein genes exhibit important tissue-dependent variation in mRNA expression. Though these genes are most stably expressed of all investigated genes in a meta-analysis they cannot be considered true reference genes

Alternative Splicing and Nonsense-Mediated RNA Decay Contribute to the Regulation of SHOX Expression

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level

Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in Drosophila

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly

Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit

Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit

Nonsense-mediated mRNA decay controls the changes in yeast ribosomal protein pre-mRNAs levels upon osmotic stress

The expression of ribosomal protein (RP) genes requires a substantial part of cellular transcription, processing and translation resources. Thus, the RP expression must be tightly regulated in response to conditions that compromise cell survival. In Saccharomyces cerevisiae cells, regulation of the RP gene expression at the transcriptional, mature mRNA stability and translational levels during the response to osmotic stress has been reported. Reprogramming global protein synthesis upon osmotic shock includes the movement of ribosomes from RP transcripts to stress-induced mRNAs. Using tiling arrays, we show that osmotic stress yields a drop in the levels of RP pre-mRNAs in S. cerevisiae cells. An analysis of the tiling array data, together with transcription rates data, shows a poor correlation, indicating that the drop in the RP pre-mRNA levels is not merely a result of the lowered RP transcription rates. A kinetic study using quantitative RT-PCR confirmed the decrease in the levels of several RP-unspliced transcripts during the first 15 minutes of osmotic stress, which seems independent of MAP kinase Hog1. Moreover, we found that the mutations in the components of the nonsense-mediated mRNA decay (NMD), Upf1, Upf2, Upf3 or in exonuclease Xrn1, eliminate the osmotic stress-induced drop in RP pre-mRNAs. Altogether, our results indicate that the degradation of yeast RP unspliced transcripts by NMD increases during osmotic stress, and suggest that this might be another mechanism to control RP synthesis during the stress response

Evolution of Alternative Splicing Regulation: Changes in Predicted Exonic Splicing Regulators Are Not Associated with Changes in Alternative Splicing Levels in Primates

Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex ‘splicing code’. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5′ splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease

Preservation of Ranking Order in the Expression of Human Housekeeping Genes

Housekeeping (HK) genes fulfill the basic needs for a cell to survive and function properly. Their ubiquitous expression, originally thought to be constant, can vary from tissue to tissue, but this variation remains largely uncharacterized and it could not be explained by previously identified properties of HK genes such as short gene length and high GC content. By analyzing microarray expression data for human genes, we uncovered a previously unnoted characteristic of HK gene expression, namely that the ranking order of their expression levels tends to be preserved from one tissue to another. Further analysis by tensor product decomposition and pathway stratification identified three main factors of the observed ranking preservation, namely that, compared to those of non-HK (NHK) genes, the expression levels of HK genes show a greater degree of dispersion (less overlap), stableness (a smaller variation in expression between tissues), and correlation of expression. Our results shed light on regulatory mechanisms of HK gene expression that are probably different for different HK genes or pathways, but are consistent and coordinated in different tissues

The Coupling of Alternative Splicing and Nonsense-Mediated mRNA Decay

Most human genes exhibit alternative splicing, but not all alternatively spliced transcripts produce functional proteins. Computational and experimental results indicate that a substantial fraction of alternative splicing events in humans result in mRNA isoforms that harbor a premature termination codon (PTC). These transcripts are predicted to be degraded by the nonsense-mediated mRNA decay (NMD) pathway. One explanation for the abundance of PTC-containing isoforms is that they represent splicing errors that are identified and degraded by the NMD pathway. Another potential explanation for this startling observation is that cells may link alternative splicing and NMD to regulate the abundance of mRNA transcripts. This mechanism, which we call "Regulated Unproductive Splicing and Translation" (RUST), has been experimentally shown to regulate expression of a wide variety of genes in many organisms from yeast to human. It is frequently employed for autoregulation of proteins that affect the splicing process itself. Thus, alternative splicing and NMD act together to play an important role in regulating gene expression