Shobha Vasudevan

Switching from repression to activation: microRNAs can up-regulate translation.

AU-rich elements (AREs) and microRNA target sites are conserved sequences in messenger RNA (mRNA) 3′ untranslated regions (3′UTRs) that control gene expression posttranscriptionally. Upon cell cycle arrest, the ARE in tumor necrosis factor–α (TNFα) mRNA is transformed into a translation activation signal, recruiting Argonaute (AGO) and fragile X mental retardation–related protein 1 (FXR1), factors associated with micro-ribonucleoproteins (microRNPs). We show that human microRNA miR369-3 directs association of these proteins with the AREs to activate translation. Furthermore, we document that two well-studied microRNAs—Let-7 and the synthetic microRNA miRcxcr4—likewise induce translation up-regulation of target mRNAs on cell cycle arrest, yet they repress translation in proliferating cells. Thus, activation is a common function of microRNPs on cell cycle arrest. We propose that translation regulation by microRNPs oscillates between repression and activation during the cell cycle.

Posttranscriptional Upregulation by MicroRNAs

MicroRNAs are small non‐coding RNA guide molecules that regulate gene expression via association with effector complexes and sequence‐specific recognition of target sites on other RNAs; misregulated microRNA expression and functions are linked to a variety of tumors, developmental disorders, and immune disease. MicroRNAs have primarily been demonstrated to mediate posttranscriptional downregulation of expression; translational repression, and deadenylation‐dependent decay of messages through partially complementary microRNA target sites in mRNA untranslated regions (UTRs). However, an emerging assortment of studies, discussed in this review, reveal that microRNAs and their associated protein complexes (microribonucleoproteins or microRNPs) can additionally function to posttranscriptionally stimulate gene expression by direct and indirect mechanisms. These reports indicate that microRNA‐mediated effects can be selective, regulated by the RNA sequence context, and associated with RNP factors and cellular conditions. Like repression, translation upregulation by microRNAs has been observed to range from fine‐tuning effects to significant alterations in expression. These studies uncover remarkable, new abilities of microRNAs and associated microRNPs in gene expression control and underscore the importance of regulation, in cis and trans, in directing appropriate microRNP responses. WIREs RNA 2012, 3:311–330. doi: 10.1002/wrna.121

Cell-cycle control of microRNA-mediated translation regulation.

MicroRNAs are small regulatory RNA molecules that exert post-transcriptional control over expression of specific target mRNAs. AU-rich elements (AREs) are highly conserved 3′UTR sequences that alter the stability and translation of mRNAs of clinical importance as a rapid and transient response to external and internal changes. We recently demonstrated that a reporter mRNA containing the tumor necrosis factor α (TNFα) ARE activates translation in response to quiescence via microRNA target sites in the ARE. Further studies revealed that microRNAs in general have the potential to regulate translation in a cell-cycle determined manner: in quiescent cells, microRNAs activate translation while in cycling/proliferating cells, microRNAs repress translation. In this study, we have analyzed microRNA regulation of translation at additional stages of the cell cycle. We observe the strongest repressive potential in the S and S/G2 phases with minimal repression in the G1 phase. Since asynchronously growing cells are predominantly in G1, these data may explain the variable magnitude of microRNA-mediated repression reported in the literature. Importantly, we observe activation in contact-inhibited G0 quiescent cells, reaffirming that the quiescent state and not serum-starvation-induced stress causes microRNA-mediated translation upregulation. In addition, we find that siRNPs, unlike microRNPs, downregulate expression of a reporter in serum-starvation-induced G0 arrested cells, as well as in proliferating cells. Our data underscore the importance of the quiescent state for microRNA-mediated translation activation and suggest the potential for further novel functions of microRNAs in distinct cell fates.

Posttranscriptional activation of gene expression in Xenopus laevis oocytes by microRNA–protein complexes (microRNPs)

MicroRNA–protein complexes (microRNPs) can activate translation of target reporters and specific mRNAs in quiescent (i.e., G0) mammalian cell lines. Induced quiescent cells, like folliculated immature oocytes, have high levels of cAMP that activate protein kinase AII (PKAII) to maintain G0 and immature states. We report microRNA-mediated up-regulated expression of reporters in immature Xenopus laevis oocytes, dependent on Xenopus AGO or human AGO2 and on FXR1, as in mammalian cells. Importantly, we find that maintenance of cAMP levels and downstream PKAII signaling are required for microRNA-mediated up-regulated expression in oocytes. We identify an important, endogenous cell state regulator, Myt1 kinase, as a natural target of microRNA-mediated up-regulation in response to xlmiR16, ensuring maintenance of oocyte immaturity. Our data reveal the physiological relevance of cAMP/PKAII-controlled posttranscriptional gene expression activation by microRNAs in maintenance of the immature oocyte state.

STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA

Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFβR2 and the TGFβ signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFβ pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.

MicroRNA-mediated mRNA translation activation in quiescent cells and oocytes involves recruitment of a nuclear microRNP.

MicroRNAs can promote translation of specific mRNAs in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes. We report that microRNA-mediated upregulation of target mRNAs in oocytes is dependent on nuclear entry of the microRNA; cytoplasmically-injected microRNA repress target mRNAs. Components of the activation microRNP, AGO, FXR1 (FXR1-iso-a) and miR16 are present in the nucleus and cytoplasm. Importantly, microRNA target mRNAs for upregulation, Myt1, TNFα and a reporter bearing the TNFα AU-rich, microRNA target sequence, are associated with AGO in immature oocyte nuclei and AGO2 in G0 human nuclei, respectively. mRNAs that are repressed or lack target sites are not associated with nuclear AGO. Crosslinking-coupled immunopurification revealed greater association of AGO2 with FXR1 in the nucleus compared to cytoplasm. Consistently, overexpression of FXR1-iso-a rescues activation of cytoplasmically-injected RNAs and in low density, proliferating cells. These data indicate the importance of a compartmentalized AGO2-FXR1-iso-a complex for selective recruitment for microRNA-mediated upregulation.

A Yeast Homologue of Hsp70, Ssa1p, Regulates Turnover of the MFA2 Transcript through Its AU-Rich 3′ Untranslated Region

ABSTRACT Many eukaryotic mRNAs exhibit regulated decay in response to cellular signals. AU-rich elements (AREs) identified in the 3′ untranslated region (3′-UTR) of several such mRNAs play a critical role in controlling the half-lives of these transcripts. The yeast ARE-containing mRNA, MFA2, has been studied extensively and is degraded by a deadenylation-dependent mechanism. However, the trans-acting factors that promote the rapid decay of MFA2 have not been identified. Our results suggest that the chaperone protein Hsp70, encoded by the SSA family of genes, is involved in modulating MFA2 mRNA decay. MFA2 is specifically stabilized in a strain bearing a temperature-sensitive mutation in the SSA1 gene. Furthermore, an AU-rich region within the 3′-UTR of the message is both necessary and sufficient to confer this regulation. Stabilization occurs as a result of slower deadenylation in the ssa1ts strain, suggesting that Hsp70 is required for activation of the turnover pathway.

p38 Mitogen-Activated Protein Kinase/Hog1p Regulates Translation of the AU-Rich-Element-Bearing MFA2 Transcript

ABSTRACT AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3′ untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3′-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3′-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3′-UTR in a regulated manner to control MFA2 3′-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3′-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3′-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3′-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.

Functional validation of microRNA-target RNA interactions.

MicroRNAs are small, non-coding RNA regulators of gene expression with important outcomes in cell state, proliferation, metabolism, immunity and development; their deregulation leads to significant clinical consequences. MicroRNAs and their associated target RNAs can be identified by genetic, bioinformatic and biochemical methods. MicroRNAs can recognize target mRNAs via direct base-pairing and recruit effector complexes to modulate their gene expression in a sequence-specific manner. MicroRNA interactions with target RNAs produce their roles in gene expression. The following are some of the validation methods employed to confirm functionally relevant microRNA interactions with their target mRNAs. Each method involves interference with the microRNA or the target mRNA to disable their interaction, which should lead to loss of microRNA-mediated gene expression if the interaction is functionally consequential. Subsequent alleviation of the interference and restoration of productive base-pairing interactions between the microRNA and target should rescue microRNA-mediated gene expression and confirm the functional requirement for direct microRNA-target mRNA interaction. Characterization of functional microRNA interactions with their target mRNAs will provide significant insights into their gene expression regulatory mechanism and lead to the development of potential therapeutic approaches to manipulate these interactions and their consequent gene expression outcomes.

Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages.

Significance This study uncovers a critical role for a general translation factor in specific developmental stages, including immature oocytes and ES cells, and during growth-factor deprivation of mammalian cells, which induces the transition to cell-cycle arrest. These conditions alter and decrease general translation yet maintain ongoing translation. We reveal upregulation of the eukaryotic translation factor 5B (eIF5B), which becomes essential for general translation, specifically in these conditions. Importantly, our data demonstrate that eIF5B controls these cell-cycle transition and developmental stages, promoting oocyte maturation and inhibiting cell-cycle arrest. These findings underscore the importance of translational regulation in cell-cycle transitions and development. Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Meti) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Meti upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Meti for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.