ABSTRACT
BACKGROUND: The speed of translation elongation is primarily determined by the abundance of tRNAs. Thus, the codon usage influences the rate with which individual mRNAs are translated. As the nature of tRNA pools and modifications can vary across biological conditions, codon elongation rates may also vary, leading to fluctuations in the protein production from individual mRNAs. Although it has been observed that functionally related mRNAs exhibit similar codon usage, presumably to provide an effective way to coordinate expression of multiple proteins, experimental evidence for codon-mediated translation efficiency modulation of functionally related mRNAs in specific conditions is scarce and the associated mechanisms are still debated. RESULTS: Here, we reveal that mRNAs whose expression increases during cell proliferation are enriched in rare codons, poorly adapted to tRNA pools. Ribosome occupancy profiling and proteomics measurements show that upon increased cell proliferation, transcripts enriched in rare codons undergo a higher translation boost than transcripts with common codons. Re-coding of a fluorescent reporter with rare codons increased protein output by ~ 30% relative to a reporter re-coded with common codons. Although the translation capacity of proliferating cells was higher compared to resting cells, we did not find evidence for the regulation of individual tRNAs. Among the models that were proposed so far to account for codon-mediated translational regulation upon changing conditions, the one that seems most consistent with our data involves a global upregulation of ready-to-translate tRNAs, which we show can lead to a higher increase in the elongation velocity at rare codons compared to common codons. CONCLUSIONS: We propose that the alleviation of translation bottlenecks in rapidly dividing cells enables preferential upregulation of pro-proliferation proteins, encoded by mRNAs that are enriched in rare codons.
Subject(s)
Cell Proliferation/genetics , Codon Usage , Peptide Chain Elongation, Translational , Animals , Mice , NIH 3T3 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
High-throughput sequencing has greatly facilitated the discovery of long and short non-coding RNAs (ncRNAs), which frequently guide ribonucleoprotein complexes to RNA targets, to modulate their metabolism and expression. However, for many ncRNAs, the targets remain to be discovered. In this study, we developed computational methods to map C/D box snoRNA target sites using data from core small nucleolar ribonucleoprotein crosslinking and immunoprecipitation and from transcriptome-wide mapping of 2Î-O-ribose methylation sites. We thereby assigned the snoRNA guide to a known methylation site in the 18S rRNA, we uncovered a novel partially methylated site in the 28S ribosomal RNA, and we captured a site in the 28S rRNA in interaction with multiple snoRNAs. Although we also captured mRNAs in interaction with snoRNAs, we did not detect 2Î-O-methylation of these targets. Our study provides an integrated approach to the comprehensive characterization of 2Î-O-methylation targets of snoRNAs in species beyond those in which these interactions have been traditionally studied and contributes to the rapidly developing field of 'epitranscriptomics'.
Subject(s)
Algorithms , High-Throughput Nucleotide Sequencing/methods , RNA, Guide, Kinetoplastida/genetics , RNA, Small Nucleolar/genetics , Ribonucleoproteins, Small Nucleolar/genetics , Transcriptome , Base Sequence , Cross-Linking Reagents/chemistry , Databases, Genetic , Immunoprecipitation , Methylation , Protein Binding , RNA, Guide, Kinetoplastida/metabolism , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Ribosomal, 28S/genetics , RNA, Ribosomal, 28S/metabolism , RNA, Small Nucleolar/metabolism , Ribonucleoproteins, Small Nucleolar/metabolism , Ribose/metabolism , SoftwareABSTRACT
Small nucleolar RNAs (snoRNAs) are a class of non-coding RNAs that guide the post-transcriptional processing of other non-coding RNAs (mostly ribosomal RNAs), but have also been implicated in processes ranging from microRNA-dependent gene silencing to alternative splicing. In order to construct an up-to-date catalog of human snoRNAs we have combined data from various databases, de novo prediction and extensive literature review. In total, we list more than 750 curated genomic loci that give rise to snoRNA and snoRNA-like genes. Utilizing small RNA-seq data from the ENCODE project, our study characterizes the plasticity of snoRNA expression identifying both constitutively as well as cell type specific expressed snoRNAs. Especially, the comparison of malignant to non-malignant tissues and cell types shows a dramatic perturbation of the snoRNA expression profile. Finally, we developed a high-throughput variant of the reverse-transcriptase-based method for identifying 2'-O-methyl modifications in RNAs termed RimSeq. Using the data from this and other high-throughput protocols together with previously reported modification sites and state-of-the-art target prediction methods we re-estimate the snoRNA target RNA interaction network. Our current results assign a reliable modification site to 83% of the canonical snoRNAs, leaving only 76 snoRNA sequences as orphan.
Subject(s)
Gene Expression Profiling , RNA Processing, Post-Transcriptional , RNA, Small Nucleolar , Transcriptome , Cluster Analysis , Computational Biology/methods , Databases, Nucleic Acid , Gene Expression Regulation , Humans , Molecular Sequence Annotation , Nucleic Acid Conformation , RNA, UntranslatedABSTRACT
BACKGROUND: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. RESULTS: Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. CONCLUSIONS: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
Subject(s)
Argonaute Proteins/metabolism , RNA, Small Nucleolar/analysis , Ribonucleoproteins, Small Nucleolar/analysis , Cross-Linking Reagents/metabolism , Genome, Human , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Models, Molecular , Molecular Sequence Data , RNA, Small Nucleolar/metabolism , Sequence Analysis, RNAABSTRACT
BACKGROUND/AIM: Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide polymorphisms (SNP) in them can have potentially wide-ranging effects. Using an association approach, this research examined the effects of the rs6505162 SNP, an A > C polymorphism located in the pre-miRNA region of miR-423, on breast cancer development. MATERIALS AND METHODS: Caucasian Australian women with breast cancer and controls matched for age and ethnicity were genotyped for rs6505162 and their genotypic and allelic frequencies analysed for significant differences. RESULTS: Analysis indicated that there were significant differences between the case and control populations (χ2 = 6.70, p = 0.035), with the CC genotype conferring reduced risk of breast cancer development (odds ratio = 0.50 95% confidence interval = 0.27-0.92, p = 0.03). CONCLUSION: Further functional research is required to determine the mechanism of action of this SNP on miRNA function.
Subject(s)
Breast Neoplasms/genetics , MicroRNAs/genetics , Case-Control Studies , Female , Genetic Predisposition to Disease , Genotype , Humans , Middle Aged , Polymorphism, Single Nucleotide , Risk FactorsABSTRACT
microRNAs are small, non-coding RNAs that influence gene expression on a post-transcriptional level. They participate in diverse biological pathways and may act as either tumor suppressor genes or oncogenes. As they may have an effect on thousands of target mRNAs, single-nucleotide polymorphisms in microRNA genes might have major functional consequences, because the microRNA's properties and/or maturation may change. miR-196a has been reported to be aberrantly expressed in breast cancer tissue. Additionally, the SNP rs11614913 in hsa-mir-196a-2 has been found to be associated with breast cancer risk in some studies although not in others. This study evaluated the association between rs11614913 and breast cancer risk in a Caucasian case-control cohort in Queensland, Australia. Results do not support an association of the tested hsa-mir-196a-2 polymorphism with breast cancer susceptibility in this cohort. As there is a discrepancy between our results and previous findings, it is important to assess the role of rs11614913 in breast cancer by further larger studies investigating different ethnic groups.