Selective probing of mRNA expression levels within a living cell.
Ontology highlight
ABSTRACT:
SUBMITTER: Nawarathna D
PROVIDER: S-EPMC2749444 | biostudies-other | 2009 Aug
REPOSITORIES: biostudies-other
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Project description:The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.
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Project description:Protein synthesis is a highly energy demanding process and is regulated according to cellular energy levels. Light and sugar availability affect mRNA translation in plant cells but the specific roles of these factors remain unclear. In this study, sucrose was applied to Arabidopsis seedlings kept in the light or in the dark, in order to distinguish sucrose and light effects on transcription and translation. These were studied using microarray analysis of steady-state mRNA and mRNA bound to translating ribosomes.Steady-state mRNA levels were affected differently by sucrose in the light and in the dark but general translation increased to a similar extent in both conditions. For a majority of the transcripts changes of the transcript levels were followed by changes in polysomal mRNA levels. However, for 243 mRNAs, a change in polysomal occupancy (defined as polysomal levels related to steady-state levels of the mRNA) was observed after sucrose treatment in the light, but not in the dark condition. Many of these mRNAs are annotated as encoding ribosomal proteins, supporting specific translational regulation of this group of transcripts. Unexpectedly, the numbers of ribosomes bound to each mRNA decreased for mRNAs with increased polysomal occupancy.Our results suggest that sucrose regulate translation of these 243 mRNAs specifically in the light, through a novel regulatory mechanism. Our data shows that increased polysomal occupancy is not necessarily leading to more ribosomes per transcript, suggesting a mechanism of translational induction not solely dependent on increased translation initiation rates.
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Project description:ObjectiveIn this study, we investigated the frequency of Epidermal growth factor receptor (EGFR) gene mutations, the level of EGFR mRNA and protein expressions in Turkish population for indicating substantial differences in the frequency of EGFR mutations, EGFR amplification and EGFR protein expression between populations and the effect of these parameters in response to EGFR tyrosine kinase inhibitors.Materials and methodsThe study included 34 patients with non-small cell lung cancers. The RNA and DNA were extracted from the normal and tumor side of the lung tissue removed by surgery. To investigate the most common mutations in the EGFR gene, exon 19 was sequenced and mutation specific PCR was performed for detecting the L858R mutation in exon 21. EGFR mRNA expression was measured by relative quantitative reverse transcription PCR. The EGFR protein levels were detected with immunohistochemistry methods from the sections of the patients' paraffin blocks.ResultsNo EGFR mutation in exon 19 or L858R mutation in exon 21 were detected in the patients. Overexpression of EGFR gene mRNA was identified in 16 of 34 (%47) patients and overexpression of EGFR protein was detected in 15 of 34 (%44) patients. Statistical analysis was not significant for the correlation between sex, age, smoking, histopathology, pathological stage and overexpression of EGFR mRNA and protein.ConclusionIt was found that in Turkish population, EGFR mutation in exon 19 and L858R mutation were very rare, EGFR protein expression was similar and EGFR mRNA expression significantly increased compared to the literature. Markedly increased EGFR mRNA expression ratios in the absence of activating mutations showed that identifying the EGFR mRNA expression level for prediction of response to EGFR tyrosine kinase inhibitors might be significant in the Turkish population.
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Project description:Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA·DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.
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Project description:Background Protein synthesis is a highly energy demanding process and is regulated according to energy availability in plant cells. Light and sugar availability affect mRNA translation but the specific roles of these factors remain unclear. In this study, sucrose was applied to Arabidopsis seedlings kept in the light or in the dark, in order to distinguish sucrose and light effects on transcription and translation. These were studied using microarray analysis of steady state mRNA and mRNA bound to translating ribosomes. Results Steady state mRNA levels were affected differently by sucrose in the light and in the dark but general translation increased to a similar extend in both conditions. Alterations in polysomal mRNA association closely followed the changes induced on the transcript level. However, for 243 mRNAs, a change in ribosome occupancy was observed after sucrose treatment in the light, but not in the dark condition. Many of these mRNAs are annotated as encoding ribosomal proteins, supporting specific translational regulation of this group of transcripts. Unexpectedly, the numbers of ribosomes bound to each mRNA decreased for mRNAs with increased ribosome occupancy. Conclusions Our results suggest that sucrose regulate translation of these 243 mRNAs but specifically in the light, through a novel regulatory mechanism. Our data sows that increased polysomal association is not necessarily leading to more ribosomes per transcript, suggesting a mechanism of translational induction not solely dependent on increased translation initiation rates. Four different samples groups were used: Control, Darkness, Sucrose, Simultaneous Darkness and sucrose treatments. All sample groups was represented by both total mRNA hybridization and of mRNAs from polysomal enrichment. All sample groups were represented by three biological replicates.
2015-01-28 | E-GEOD-59306 | biostudies-arrayexpress
Project description:Background Protein synthesis is a highly energy demanding process and is regulated according to energy availability in plant cells. Light and sugar availability affect mRNA translation but the specific roles of these factors remain unclear. In this study, sucrose was applied to Arabidopsis seedlings kept in the light or in the dark, in order to distinguish sucrose and light effects on transcription and translation. These were studied using microarray analysis of steady state mRNA and mRNA bound to translating ribosomes. Results Steady state mRNA levels were affected differently by sucrose in the light and in the dark but general translation increased to a similar extend in both conditions. Alterations in polysomal mRNA association closely followed the changes induced on the transcript level. However, for 243 mRNAs, a change in ribosome occupancy was observed after sucrose treatment in the light, but not in the dark condition. Many of these mRNAs are annotated as encoding ribosomal proteins, supporting specific translational regulation of this group of transcripts. Unexpectedly, the numbers of ribosomes bound to each mRNA decreased for mRNAs with increased ribosome occupancy. Conclusions Our results suggest that sucrose regulate translation of these 243 mRNAs but specifically in the light, through a novel regulatory mechanism. Our data sows that increased polysomal association is not necessarily leading to more ribosomes per transcript, suggesting a mechanism of translational induction not solely dependent on increased translation initiation rates.
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