Final Report Summary - MIREG (Identifying novel regulatory mechanisms of miRNA functions)
Achievements made by this grant
Major role for mRNA stability in shaping the kinetics of gene induction
mRNA levels in cells are determined by the relative rates of RNA production and degradation. Yet, to date, most analyses of gene expression activation were focused on mechanisms that regulate transcription, while the role of mRNA stability in modulating transcriptional networks was to a large extent overlooked. We examined on a global scale the role of mRNA stability in shaping the kinetics of gene response, and revealed a striking global anti-correlation between rapidity of induction and mRNA stability, fitting the prediction of a kinetic mathematical model. Our results demonstrate the key role of mRNA stability in determining induction kinetics in mammalian transcriptional networks.
Bioinformatics approaches to uncover miRNA activity from expression data sets.
Elucidation of regulatory roles played by miRNAs in various biological networks is one of the greatest challenges of present molecular and computational biology. The integrated analysis of gene expression data and 3'-UTR sequences holds great promise for being an effective mean to systematically delineate active miRNAs in different biological processes.
Applying such an integrated analysis, we show that in the majority of current arrays probes are selected from target transcript 3'-UTRs. We developed visualization and normalization schemes for the detection and removal of such biases, and demonstrate that their application to microarray data significantly enhances the computational identification of active miRNAs.
Control of miRNA activity by RNA binding proteins in development, cellular stress response, and cancer.
The evolutionary conservation of 3’UTRs is not restricted to the miRNA-targeting sequences, but often includes regions with high conservation, suggesting that other factors (proteins or RNA) could associate with this region to influence miRNA-mRNA function and/or interaction. To investigate this possibility we developed a reporter assay to screen an overexpression library for RBPs that influence miRNA function. This has resulted in few published papers and one that is now under revision. In short:
a) We found that human dead end 1 (DND1), a protein whose function is required for germ cell survival and migration in zebrafish, inhibits miRNA activity of target mRNAs (Kedde et al 2007). Specifically, in primordial germ cells, DND1 protects the expression of several genes from repression by miRNAs.
b) We uncovered a novel RBP-induced structural switch that controls microRNA-mediated gene expression regulation (Kedde et al., 2010). In particular, the Pumilio RNA binding protein induces an RNA structure switch in p27-3ʹ UTR that controls miR-221 and miR-222 accessibility and the cellular response to growth factors.
c) We show that selective inhibition of miRNA accessibility by RBM38 is required for the p53 tumor suppressor activity. This is particularly interesting as cancers characterized by wild type p53 frequently tend to inhibit rbm38 expression by promoter methylation, suggesting that inhibition of rbm38 is a way to turn normal cells cancer while leaving p53 intact.
d) Recently we summarized latest developments in the field of miRNA regulation by RNA binding proteins in cancer, in the form of a review entitled “MicroRNA regulation by RNA binding proteins and its implications for cancer” which will be published later this year in the top review journal Nature Cancer Reviews (van Kouwenhove et al.,). This will guarantee dissemination of our achievements and knowledge to the entire scientific community.
Regulation of alternative cleavage and polyadenylation of mRNAs
The 3' end of most protein-coding genes and long non-coding RNAs is cleaved and polyadenylated. Recent discoveries have revealed that a large proportion of these genes contains more than one polyadenylation site. Therefore, alternative polyadenylation (APA) is a widespread phenomenon, generating mRNAs with alternative 3' ends. APA contributes to the complexity of the transcriptome by generating isoforms that differ either in their coding sequence or in their 3' untranslated regions (UTRs), thereby potentially regulating the function, stability, localization and translation efficiency of target RNAs. We recently discovered extensive APA alterations associated with the gene PABPN1, the causal agent of the genetic disease OPMD (ocular pharyngeal muscular dystrophy). In particular, the disease-mutant PABPN1 functions in a dominant negative manner to induce the usage of alternative weak polyadenylation sites that are closer to the transcription start site. Our results link for the first time APA with a genetic disorder.
In addition, we identified extensive alterations in APA occurring during the transition of cells from arrested to proliferative and transformed states. Interestingly, a significantly enhanced cleavage occurred at sites located in coding sequence or within introns, consequently generating truncated mRNAs with the potential production of aberrant proteins. These aberrant proteins carry the potential to affect cell behavior by either acting in a dominant negative manner, gain additional functions, or lose any function. Currently we examine causal effects of APA in cancer.
Role of enhancer RNAs in controlling gene expression and in cancer
It is well known that the p53 tumor suppressor gene regulates transcription and cell cycle progression by binding within or nearby target genes controlling cell proliferation and survival. We found that p53 binds genomic regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound regions are indeed enhancers. Moreover, these p53-binding sites produce enhancer RNAs (eRNAs) that are required for enhancer activity. The production of eRNAs is interesting, as they can be used to control enhancer activity and phenotype
Major role for mRNA stability in shaping the kinetics of gene induction
mRNA levels in cells are determined by the relative rates of RNA production and degradation. Yet, to date, most analyses of gene expression activation were focused on mechanisms that regulate transcription, while the role of mRNA stability in modulating transcriptional networks was to a large extent overlooked. We examined on a global scale the role of mRNA stability in shaping the kinetics of gene response, and revealed a striking global anti-correlation between rapidity of induction and mRNA stability, fitting the prediction of a kinetic mathematical model. Our results demonstrate the key role of mRNA stability in determining induction kinetics in mammalian transcriptional networks.
Bioinformatics approaches to uncover miRNA activity from expression data sets.
Elucidation of regulatory roles played by miRNAs in various biological networks is one of the greatest challenges of present molecular and computational biology. The integrated analysis of gene expression data and 3'-UTR sequences holds great promise for being an effective mean to systematically delineate active miRNAs in different biological processes.
Applying such an integrated analysis, we show that in the majority of current arrays probes are selected from target transcript 3'-UTRs. We developed visualization and normalization schemes for the detection and removal of such biases, and demonstrate that their application to microarray data significantly enhances the computational identification of active miRNAs.
Control of miRNA activity by RNA binding proteins in development, cellular stress response, and cancer.
The evolutionary conservation of 3’UTRs is not restricted to the miRNA-targeting sequences, but often includes regions with high conservation, suggesting that other factors (proteins or RNA) could associate with this region to influence miRNA-mRNA function and/or interaction. To investigate this possibility we developed a reporter assay to screen an overexpression library for RBPs that influence miRNA function. This has resulted in few published papers and one that is now under revision. In short:
a) We found that human dead end 1 (DND1), a protein whose function is required for germ cell survival and migration in zebrafish, inhibits miRNA activity of target mRNAs (Kedde et al 2007). Specifically, in primordial germ cells, DND1 protects the expression of several genes from repression by miRNAs.
b) We uncovered a novel RBP-induced structural switch that controls microRNA-mediated gene expression regulation (Kedde et al., 2010). In particular, the Pumilio RNA binding protein induces an RNA structure switch in p27-3ʹ UTR that controls miR-221 and miR-222 accessibility and the cellular response to growth factors.
c) We show that selective inhibition of miRNA accessibility by RBM38 is required for the p53 tumor suppressor activity. This is particularly interesting as cancers characterized by wild type p53 frequently tend to inhibit rbm38 expression by promoter methylation, suggesting that inhibition of rbm38 is a way to turn normal cells cancer while leaving p53 intact.
d) Recently we summarized latest developments in the field of miRNA regulation by RNA binding proteins in cancer, in the form of a review entitled “MicroRNA regulation by RNA binding proteins and its implications for cancer” which will be published later this year in the top review journal Nature Cancer Reviews (van Kouwenhove et al.,). This will guarantee dissemination of our achievements and knowledge to the entire scientific community.
Regulation of alternative cleavage and polyadenylation of mRNAs
The 3' end of most protein-coding genes and long non-coding RNAs is cleaved and polyadenylated. Recent discoveries have revealed that a large proportion of these genes contains more than one polyadenylation site. Therefore, alternative polyadenylation (APA) is a widespread phenomenon, generating mRNAs with alternative 3' ends. APA contributes to the complexity of the transcriptome by generating isoforms that differ either in their coding sequence or in their 3' untranslated regions (UTRs), thereby potentially regulating the function, stability, localization and translation efficiency of target RNAs. We recently discovered extensive APA alterations associated with the gene PABPN1, the causal agent of the genetic disease OPMD (ocular pharyngeal muscular dystrophy). In particular, the disease-mutant PABPN1 functions in a dominant negative manner to induce the usage of alternative weak polyadenylation sites that are closer to the transcription start site. Our results link for the first time APA with a genetic disorder.
In addition, we identified extensive alterations in APA occurring during the transition of cells from arrested to proliferative and transformed states. Interestingly, a significantly enhanced cleavage occurred at sites located in coding sequence or within introns, consequently generating truncated mRNAs with the potential production of aberrant proteins. These aberrant proteins carry the potential to affect cell behavior by either acting in a dominant negative manner, gain additional functions, or lose any function. Currently we examine causal effects of APA in cancer.
Role of enhancer RNAs in controlling gene expression and in cancer
It is well known that the p53 tumor suppressor gene regulates transcription and cell cycle progression by binding within or nearby target genes controlling cell proliferation and survival. We found that p53 binds genomic regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound regions are indeed enhancers. Moreover, these p53-binding sites produce enhancer RNAs (eRNAs) that are required for enhancer activity. The production of eRNAs is interesting, as they can be used to control enhancer activity and phenotype