Xinjun Ji

MicroRNA silencing through RISC recruitment of eIF6

MicroRNAs (miRNAs) are a class of small RNAs that act post-transcriptionally to regulate messenger RNA stability and translation. To elucidate how miRNAs mediate their repressive effects, we performed biochemical and functional assays to identify new factors in the miRNA pathway. Here we show that human RISC (RNA-induced silencing complex) associates with a multiprotein complex containing MOV10—which is the homologue of Drosophila translational repressor Armitage—and proteins of the 60S ribosome subunit. Notably, this complex contains the anti-association factor eIF6 (also called ITGB4BP or p27BBP), a ribosome inhibitory protein known to prevent productive assembly of the 80S ribosome. Depletion of eIF6 in human cells specifically abrogates miRNA-mediated regulation of target protein and mRNA levels. Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of the endogenous LIN-14 and LIN-28 target protein and mRNA levels. These results uncover an evolutionarily conserved function of the ribosome anti-association factor eIF6 in miRNA-mediated post-transcriptional silencing.

Hypoxia-mediated Selective mRNA Translation by an Internal Ribosome Entry Site-independent Mechanism

Although it is advantageous for hypoxic cells to inhibit protein synthesis and conserve energy, it is also important to translate mRNAs critical for adaptive responses to hypoxic stress. Because internal ribosome entry sites (IRES) have been postulated to mediate this preferential synthesis, we analyzed the 5 ′-untranslated regions from a panel of stress-regulated mRNAs for m7GTP cap-independent translation and identified putative IRES elements in encephalomyocarditis virus, vascular endothelial growth factor, hypoxia-inducible factors (HIFs) 1α and 2α, glucose transporter-like protein 1, p57Kip2, La, BiP, and triose phosphate isomerase transcripts. However, when capped and polyadenylated dicistronic RNAs were synthesized in vitro and transfected into cells, cellular IRES-mediated translation accounted for less than 1% that of the level of cap-dependent translation. Moreover, hypoxic stress failed to activate cap-independent synthesis, indicating that it is unlikely that this is the primary mechanism for the maintenance of the translation of these mRNAs under low O2. Furthermore, although HIF-1α is frequently cited as an example of an mRNA that is preferentially translated, we demonstrate that under different levels and durations of hypoxic stress, changes in newly synthesized HIF-1α and β-actin protein levels mirror alterations in corresponding mRNA abundance. In addition, our data suggest that cyclin-dependent kinase inhibitor p57Kip2 and vascular endothelial growth factor mRNAs are selectively translated by an IRES-independent mechanism under hypoxic stress.

In Vivo Association of the Stability Control Protein αCP with Actively Translating mRNAs

ABSTRACT Posttranscriptional controls play a major role in eucaryotic gene expression. These controls are mediated by sequence-specific interactions of cis-acting determinants in target mRNAs with one or more protein factors. The positioning of a subset of these mRNA-protein (RNP) complexes within the 3′ untranslated region (3′ UTR) may allow them to remain associated with the mRNA during active translation. Robust expression of human α-globin mRNA during erythroid differentiation has been linked to formation of a binary complex between a KH-domain protein, αCP, and a 3′ UTR C-rich motif. Detection of this “α-complex” has been limited to in vitro studies, and the functional state of the α-globin mRNA targeted by αCP has not been defined. In the present study we demonstrate that a significant fraction of αCP is associated with polysomal mRNA. Targeted analysis of the polysomal RNP complexes revealed that αCP is specifically bound to actively translating α-globin mRNA. The bound αCP is restricted to the poly(C)-rich 3′ UTR motif and is dislodged when ribosomes are allowed to enter this region. These data validate the general importance of the 3′ UTR as a sheltered site for RNP complexes and support a specific model in which the stabilizing function of αCP is mediated on actively translating target mRNAs.

The KH-Domain Protein αCP Has a Direct Role in mRNA Stabilization Independent of Its Cognate Binding Site

ABSTRACT Previous studies suggest that high-level stability of a subset of mammalian mRNAs is linked to a C-rich motif in the 3′ untranslated region (3′UTR). High-level expression of human α-globin mRNA (hα-globin mRNA) in erythroid cells has been specifically attributed to formation of an RNA-protein complex comprised of a 3′UTR C-rich motif and an associated 39-kDa poly(C) binding protein, αCP. Documentation of this RNA-protein α-complex has been limited to in vitro binding studies, and its impact has been monitored by alterations in steady-state mRNA. Here we demonstrate that αCP is stably bound to hα-globin mRNA in vivo, that α-complex assembly on the hα-globin mRNA is restricted to the 3′UTR C-rich motif, and that α-complex assembly extends the physical half-life of hα-globin mRNA selectively in erythroid cells. Significantly, these studies also reveal that an artificially tethered αCP has the same mRNA-stabilizing activity as the native α-complex. These data demonstrate a unique contribution of the α-complex to hα-globin mRNA stability and support a model in which the sole function of the C-rich motif is to selectively tether αCP to a subset of mRNAs. Once bound, αCP appears to be fully sufficient to trigger downstream events in the stabilization pathway.

The 3′ Untranslated Region Complex Involved in Stabilization of Human α-globin mRNA Assembles in the Nucleus and Serves an Independent Role as a Splice Enhancer

ABSTRACT Posttranscriptional controls, mediated primarily by RNA-protein complexes, have the potential to alter multiple steps in RNA processing and function. Human α-globin mRNA is bound at a C-rich motif in the 3′ untranslated region (3′UTR) by the KH domain protein α-globin poly(C)-binding protein (αCP). This “α-complex” is essential to cytoplasmic stability of α-globin mRNA in erythroid cells. Here we report that the 3′UTR α-complex also serves an independent nuclear role as a splice enhancer. Consistent with this role, we find that αCP binds α-globin transcripts prior to splicing. Surprisingly, this binding occurs at C-rich sites within intron I as well as at the 3′UTR C-rich determinant. The intronic and 3′UTR αCP complexes appear to have distinct effects on splicing. While intron I complexes repress intron I excision, the 3′UTR complex enhances splicing of the full-length transcript both in vivo and in vitro. In addition to its importance to splicing, nuclear assembly of the 3′UTR αCP complex may serve to “prepackage” α-globin mRNA with its stabilizing complex prior to cytoplasmic export. Linking nuclear and cytoplasmic controls by the action of a particular RNA-binding protein, as reported here, may represent a modality of general importance in eukaryotic gene regulation.

αCP Poly(C) Binding Proteins Act as Global Regulators of Alternative Polyadenylation

ABSTRACT We have previously demonstrated that the KH-domain protein αCP binds to a 3′ untranslated region (3′UTR) C-rich motif of the nascent human alpha-globin (hα-globin) transcript and enhances the efficiency of 3′ processing. Here we assess the genome-wide impact of αCP RNA-protein (RNP) complexes on 3′ processing with a specific focus on its role in alternative polyadenylation (APA) site utilization. The major isoforms of αCP were acutely depleted from a human hematopoietic cell line, and the impact on mRNA representation and poly(A) site utilization was determined by direct RNA sequencing (DRS). Bioinformatic analysis revealed 357 significant alterations in poly(A) site utilization that could be specifically linked to the αCP depletion. These APA events correlated strongly with the presence of C-rich sequences in close proximity to the impacted poly(A) addition sites. The most significant linkage was the presence of a C-rich motif within a window 30 to 40 bases 5′ to poly(A) signals (AAUAAA) that were repressed upon αCP depletion. This linkage is consistent with a general role for αCPs as enhancers of 3′ processing. These findings predict a role for αCPs in posttranscriptional control pathways that can alter the coding potential and/or levels of expression of subsets of mRNAs in the mammalian transcriptome.

An RNA–protein complex links enhanced nuclear 3′ processing with cytoplasmic mRNA stabilization

Post‐transcriptional controls are critical to gene regulation. These controls are frequently based on sequence‐specific binding of trans‐acting proteins to cis‐acting motifs on target RNAs. Prior studies have revealed that the KH‐domain protein, αCP, binds to a 3′ UTR C‐rich motif of hα‐globin mRNA and contributes to its cytoplasmic stability. Here, we report that this 3′ UTR αCP complex regulates the production of mature α‐globin mRNA by enhancing 3′ processing of the hα‐globin transcript. We go on to demonstrate that this nuclear activity reflects enhancement of both the cleavage and the polyadenylation reactions and that αCP interacts in vivo with core components of the 3′ processing complex. Consistent with its nuclear processing activity, our studies reveal that αCP assembles co‐transcriptionally at the hα‐globin chromatin locus and that this loading is selectively enriched at the 3′ terminus of the gene. The demonstrated linkage of nuclear processing with cytoplasmic stabilization via a common RNA–protein complex establishes a basis for integration of sequential controls critical to robust and sustained expression of a target mRNA.

Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis

Insulin‐like growth factor binding protein (IGFBP)‐3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia‐inducible factor (HIF)‐1α, but not HIF‐2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF‐1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 κb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia‐mediated inhibition of global protein synthesis. 7‐Methyl GTP‐cap binding assays suggested that hypoxia suppresses cap‐dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap‐dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap‐independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF‐1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein‐Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF‐1α‐mediated mRNA transcription and continuous protein synthesis. FASEB J. 26, 2620‐2630 (2012). www.fasebj.org

αCP binding to a cytosine-rich subset of polypyrimidine tracts drives a novel pathway of cassette exon splicing in the mammalian transcriptome

Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5′ to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5′ to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome.

Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells

A CRISPR screen for RBC regulators Hemoglobin in red blood cells (RBCs) carries oxygen to the tissues. Sickle cell disease is an inherited condition that involves abnormal hemoglobin. Current treatments entail modulating the level of fetal hemoglobin expression. Grevet et al. performed a CRISPR-Cas9 screen for regulators of fetal hemoglobin in RBCs and identified heme-regulated eIF2α kinase (HRI). Depleting the kinase in RBCs led to an increase in fetal hemoglobin levels and reduced sickling of cultured human RBCs. Thus, HRI may be a therapeutic target for sickle cell disease and other hemoglobin disorders. Science, this issue p. 285 HRI kinase represses expression of fetal hemoglobin and provides a potential target for sickle cell disease treatment. Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of β-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain–focused CRISPR-Cas9–based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.