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ABSTRACT:
PROVIDER: PRJNA1276515 | ENA |
REPOSITORIES: ENA
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Project description:The mRNA 5â² cap is normally essential for eukaryotic mRNA translation, stabilization and transport and both the cap and eIF4E are important elements of post-transcriptional gene regulation. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite translation initiation factor eIF4E and its interaction with 5â² capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA 5â² cap binding activity. We used this protein to purify P. falciparum full-length 5â² capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified 5â² capped mRNAs shows that a subset of 34 features were more than two-fold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal. Keywords: total RNA vs purified capped mRNA The microarray data were obtained from four hybridizations using RNA from two independent GST-PfeIF4E purifications from separate malaria cultures.
2010-06-10 | E-GEOD-6356 | biostudies-arrayexpress
Project description:The mRNA 5′ cap is normally essential for eukaryotic mRNA translation, stabilization and transport and both the cap and eIF4E are important elements of post-transcriptional gene regulation. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite translation initiation factor eIF4E and its interaction with 5′ capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA 5′ cap binding activity. We used this protein to purify P. falciparum full-length 5′ capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified 5′ capped mRNAs shows that a subset of 34 features were more than two-fold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal. Keywords: total RNA vs purified capped mRNA
2007-07-07 | GSE6356 | GEO
Project description:Nuclear mRNA processing occurs in a stepwise manner to generate the protein blueprints required for cellular function. The addition of the methyl-7-guanosine (m7G) cap on the 5’end of mRNAs is conserved in humans, plants and fungi. The m7G cap recruits the nuclear cap-binding protein NCBP2 co-/peri-transcriptionally where it mediates interactions between capped-RNAs and the processing machinery. Convention posits that NCBP2 is the sole cap-binding protein in the nucleus positioning cap-chaperoning as an important constitutive, housekeeping function. However, the eukaryotic translation initiation factor eIF4E is also found in the nucleus across Kingdoms. Both eIF4E and NCBP2 employ highly similar structural strategies to directly bind the m7G cap. The presence of two cap-binding factors in the nucleus could arise to provide redundancy to ensure gene expression security or it could provide the basis for tandem regulation of selected subsets of capped-mRNAs driving distinct gene expression programmes. To dissect these possibilities, we compared the spatial localizations, interactomes and impacts on gene expression of eIF4E and NCBP2. We found that like NCBP2, eIF4E physically and spatially associated with active sites of transcription and splicing machinery. However, contrary to expectations, eIF4E and NCBP2 drove distinct transcription and splicing signatures impacting ~1000 transcripts which in turn elicit different biological programmes. RNAs segregated with specific cap-binding proteins with only an overlap of only ~130 transcripts between eIF4E and NCBP2. Clearly, the cap-interaction was not sufficient to elicit sensitivity to these cap-binding proteins given the absence of generalized impacts on the transcriptome. We uncovered the molecular mechanism for cap-chaperone selectivity which lay in differences in conserved sequences motifs within introns of selected mRNAs, their mainly distinct spatial localizations, and differences in the capacity to alter splice factor production. Relevant to the 130 common targets, a fraction of eIF4E and NCBP2 did interact and spatially overlap. Despite their common m7G cap-binding activity, eIF4E and NCBP2 elicit substantively different impacts on gene expression. In contrast to the conventional paradigm, our data support a model whereby cap-chaperones direct processing of distinct subsets of mRNAs thereby eliciting diverse biological programmes. Differential programming by cap-chaperones reveals an unexpected regulatory point in gene expression..
2025-08-13 | GSE295397 | GEO
Project description:Nuclear mRNA processing occurs in a stepwise manner to generate the protein blueprints required for cellular function. The addition of the methyl-7-guanosine (m7G) cap on the 5’end of mRNAs is conserved in humans, plants and fungi. The m7G cap recruits the nuclear cap-binding protein NCBP2 co-/peri-transcriptionally where it mediates interactions between capped-RNAs and the processing machinery. Convention posits that NCBP2 is the sole cap-binding protein in the nucleus positioning cap-chaperoning as an important constitutive, housekeeping function. However, the eukaryotic translation initiation factor eIF4E is also found in the nucleus across Kingdoms. Both eIF4E and NCBP2 employ highly similar structural strategies to directly bind the m7G cap. The presence of two cap-binding factors in the nucleus could arise to provide redundancy to ensure gene expression security or it could provide the basis for tandem regulation of selected subsets of capped-mRNAs driving distinct gene expression programmes. To dissect these possibilities, we compared the spatial localizations, interactomes and impacts on gene expression of eIF4E and NCBP2. We found that like NCBP2, eIF4E physically and spatially associated with active sites of transcription and splicing machinery. However, contrary to expectations, eIF4E and NCBP2 drove distinct transcription and splicing signatures impacting ~1000 transcripts which in turn elicit different biological programmes. RNAs segregated with specific cap-binding proteins with only an overlap of only ~130 transcripts between eIF4E and NCBP2. Clearly, the cap-interaction was not sufficient to elicit sensitivity to these cap-binding proteins given the absence of generalized impacts on the transcriptome. We uncovered the molecular mechanism for cap-chaperone selectivity which lay in differences in conserved sequences motifs within introns of selected mRNAs, their mainly distinct spatial localizations, and differences in the capacity to alter splice factor production. Relevant to the 130 common targets, a fraction of eIF4E and NCBP2 did interact and spatially overlap. Despite their common m7G cap-binding activity, eIF4E and NCBP2 elicit substantively different impacts on gene expression. In contrast to the conventional paradigm, our data support a model whereby cap-chaperones direct processing of distinct subsets of mRNAs thereby eliciting diverse biological programmes. Differential programming by cap-chaperones reveals an unexpected regulatory point in gene expression..
2025-08-13 | GSE300496 | GEO
Project description:Translation factors eIF4E and eIF4G form eIF4F, which binds to mRNAs to promote ribosome recruitment and translation initiation. We were interested in analysing the effects of stresses on eIF4F interactions with individual mRNAs. We used a RIP-seq approach to assess how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change in response to three stresses: 1) addition of hydrogen peroxide; 2) amino acids withdrawal; and, 3) glucose withdrawal. We find that acute stress leads to changes in eIF4FmRNA interactions that are shared among each factor and across the stresses imposed.
2017-10-11 | E-MTAB-5836 | biostudies-arrayexpress
Project description:Here we develop a novel methodology, capCLIP, to capture and identify mRNA interactions with the major cellular cap-binding protein eIF4E.
2020-12-31 | GSE138473 | GEO
Project description:Eukaryotic mRNA 5’ end is decorated with cap structure which plays multiple roles in the cell. Most importantly, protects transcript from exonucleolytic degradation and enables translation of encoded protein conducted by cap-dependent machinery. This is also a key feature in the process of recognizing RNA as self or non-self molecule by cellular innate immune system. Cap is composed of 7-methylguanosine linked by a 5′,5′-triphosphate chain to the first transcribed nucleotide, and is formed enzymatically during transcription. This m7GpppN structure, called cap0, can be further modified by CMTR1 methyltransferase to cap1 (m7GpppNm), which predominates in eukaryotic cells. Cap2, with additional 2′-O-methylation at the second transcribed nucleotide (m7GpppNmpNm) added by another methyltransferase CMTR2, also can be found at mRNA 5’ end. We present new tools allowing for in vitro synthesis of RNAs possessing cap2, i.e tetranucleotide cap analogues. Utilizing these for in vitro transcription reactions we obtained RNAs with 2′-O-methylation present at the second transcribed nucleotide. Due to that we investigated the role of cap2 in protein production from reporter mRNA protected with this structure in different conditions, normal or stress ones, or with CMTR1/2 down regulated level. We also assessed the affinity of eIF4E protein to differentially capped RNAs. Furthermore, we verified whether an additional 2′-O-methylation presence in capped RNA makes transcript more resistant to decapping enzyme action.
2022-09-08 | PXD028635 | Pride
Project description:Eukaryotic mRNA 5’ end is decorated with cap structure which plays multiple roles in the cell. Most importantly, protects transcript from exonucleolytic degradation and enables translation of encoded protein conducted by cap-dependent machinery. This is also a key feature in the process of recognizing RNA as self or non-self molecule by cellular innate immune system. Cap is composed of 7-methylguanosine linked by a 5′,5′-triphosphate chain to the first transcribed nucleotide, and is formed enzymatically during transcription. This m7GpppN structure, called cap0, can be further modified by CMTR1 methyltransferase to cap1 (m7GpppNm), which predominates in eukaryotic cells. Cap2, with additional 2′-O-methylation at the second transcribed nucleotide (m7GpppNmpNm) added by another methyltransferase CMTR2, also can be found at mRNA 5’ end. We present new tools allowing for in vitro synthesis of RNAs possessing cap2, i.e tetranucleotide cap analogues. Utilizing these for in vitro transcription reactions we obtained RNAs with 2′-O-methylation present at the second transcribed nucleotide. Due to that we investigated the role of cap2 in protein production from reporter mRNA protected with this structure in different conditions, normal or stress ones, or with CMTR1/2 down regulated level. We also assessed the affinity of eIF4E protein to differentially capped RNAs. Furthermore, we verified whether an additional 2′-O-methylation presence in capped RNA makes transcript more resistant to decapping enzyme action.
2022-09-08 | PXD028636 | Pride
Project description:Recent studies of the human transcriptome suggest that many transcripts have short or lack 3' poly(A) ends. In addition, the length of 3' poly(A) ends may vary for some transcript to regulate mRNA translation. Heterogeneous lengths of poly(A) ends may introduce variations in measuring RNA transcripts using current approaches. We hypothesized that affinity purifying Pol II RNAs by their unique 5' m7GpppN cap regardless of their polyadenylation status would add additional information to genomic expression analyses and possibly overcome the variation in signals sometimes observed with analysis of poly(A) selected mRNA. 5' capped and 3' poly(A) RNA were isolated from normal and hepatitis C cirrhotic (HCV) human liver using a high-affinity variant of eIF4E and standard oligo(dT) methods, respectively. Both populations of RNA were analyzed using ENCODE tiling arrays representing 1% of the genome. A total of 64 annotated genes were significantly increased in HCV as compared to normal liver; 27 of these genes were identified by analyzing 5' capped RNA. A total of 31 annotated genes were significantly decreased; 16 of these were identified by analyzing 5' capped RNA. Bioinformatic analysis showed that 5' capped RNA provided a more extensive expression profile of intronic regions and identified Pol II transcriptionally active regions in unannotated regions of the genome that were differentially expressed in normal and disease states. The analysis of 5' capped RNA isolated from biospecimens provides additional gene expression information and identifies novel Pol II transcripts that are differentially expressed. This approach may also be useful in studying of RNAs regulated by 3' polyadenylation or RNA degradation. Four-condition experiment: 5'Cap and 3'Poly(A) isolation in both Normal and HCV Cirrhotic liver. Biological samples: 1 experimental, 1 control. Experimental replicates: 4 of each sample, for a total of 16 samples.
2011-03-29 | E-GEOD-18904 | biostudies-arrayexpress
Project description:Recent studies of the human transcriptome suggest that many transcripts have short or lack 3' poly(A) ends. In addition, the length of 3' poly(A) ends may vary for some transcript to regulate mRNA translation. Heterogeneous lengths of poly(A) ends may introduce variations in measuring RNA transcripts using current approaches. We hypothesized that affinity purifying Pol II RNAs by their unique 5' m7GpppN cap regardless of their polyadenylation status would add additional information to genomic expression analyses and possibly overcome the variation in signals sometimes observed with analysis of poly(A) selected mRNA. 5' capped and 3' poly(A) RNA were isolated from normal and hepatitis C cirrhotic (HCV) human liver using a high-affinity variant of eIF4E and standard oligo(dT) methods, respectively. Both populations of RNA were analyzed using ENCODE tiling arrays representing 1% of the genome. A total of 64 annotated genes were significantly increased in HCV as compared to normal liver; 27 of these genes were identified by analyzing 5' capped RNA. A total of 31 annotated genes were significantly decreased; 16 of these were identified by analyzing 5' capped RNA. Bioinformatic analysis showed that 5' capped RNA provided a more extensive expression profile of intronic regions and identified Pol II transcriptionally active regions in unannotated regions of the genome that were differentially expressed in normal and disease states. The analysis of 5' capped RNA isolated from biospecimens provides additional gene expression information and identifies novel Pol II transcripts that are differentially expressed. This approach may also be useful in studying of RNAs regulated by 3' polyadenylation or RNA degradation.
2011-03-29 | GSE18904 | GEO