Unraveling cis-regulatory elements by mapping structural changes in mRNAs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE102069 | GEO | 2018/10/03
REPOSITORIES: GEO
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Project description:Translational dysregulation is an emerging hallmark of cancer, and increased activity of the mRNA helicase eIF4A is associated with poor survival in malignancies. This is believed to be due to the unwinding of secondary structures within the 5’UTRs of oncogenic mRNAs, with studies showing that in general eIF4A-dependent mRNAs have longer 5’UTRs with more stable secondary structures, yet our ability to predict eIF4A-dependency from 5’UTR properties alone remains poor. We therefore used Structure-seq 2 to measure transcriptome-wide changes in RNA structure in MCF7 cells, following eIF4A inhibition with hippuristanol. This technique measures the single-strandedness of RNA by specific and rapid methylation of single-stranded adenosines and cytosines with Dimethyl Sulphate (DMS). When paired with polysome profiling data to identify which mRNAs are most translationally repressed, we can identify the structural determinants of eIF4A-dependency. Upon eIF4A inhibition, both 5’UTRs and CDSs become generally more structured, while overall this was not observed for 3’UTRs. This was most pronounced in CDSs, supporting recent findings that the ribosome sculpts RNA structure in this region. 5’UTRs are generally more structured at their 5’ ends and highly translated mRNAs are less structured just upstream of the CDS. Following eIF4A inhibition, the 5’UTR is remodelled. eIF4A-dependent mRNAs have greater localised gains of structure. The degree of these structural changes is strongly correlated with 5’UTR length, explaining why eIF4A-dependent mRNAs have longer 5’UTRs. Crucially, in eIF4A-dependent mRNAs these highly-structured elements are located predominantly at the 3’ end of the 5’UTR, suggesting that increased structure just upstream of the CDS is most inhibitory to translation following eIF4A inhibition and is a key determinant of eIF4A-dependency.
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Project description:Firczuk2013 - Eukaryotic mRNA translation machinery
This is a model of Saccharomyces cerevisiae
mRNA translation which includes the initiation, elongation and termination phases. The model is for 20 condon mRNAs. The building of a multi-factor complex in initiation and also the different processes in elongation and termination are modelled in detail. The model takes into account that ribosomes cover more than one codon of mRNA so that the movement of ribosomes are effectively blocked by other ribosomes several codons downstream. It is assumed that 15 codons are occupied by each ribosome. This blocking effect is considered in reaction R18 in initiation and also reaction R26, the reaction where translocation of ribosomes takes place in elongation. The kinetic functions of these two reactions are based on MacDonald et al. 1968 and Heinrich & Rapaport 1980. All other kinetic functions follow mass-action kinetics. The concentrations of transfer RNA species (Met-tRNA, aa-tRNA and tRNA in the model) are kept constant, while the other species' concentrations can change in the course of the simulation. The model describes the translation of a short mRNA with 20 codons. Therefore, all reactions in the elongation cycle (R22, R23, R25, R26, R28 and R29) and the corresponding species are replicated accordingly to model the species with ribosomes bound at different positions. In summary, the model contains 165 different species and 141 reactions.
The value of the 56 rate constant parameters were estimated by fitting the model against a series of experimental data consisting of modulation of the various translation factors (Figures 2, 3 and S3). Overall the parameter estimation was carried out over 212 different data points (steady states).
This model is described in the article:
An in vivo control map for the eukaryotic mRNA translation machinery
Helena Firczuk, Shichina Kannambath, Jürgen Pahle, Amy Claydon, Robert Beynon, John Duncan, Hans Westerhoff, Pedro Mendes and John EG McCarthy
Molecular Systems Biology. 9:635
Abstract:
Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.
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2011-04-30 | BIOMD0000000457 | BioModels
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Project description:Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.
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