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.

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.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50 -54 nucleotides 5 to the final exon-exon junction. We have described a set of naturally occurring human ␤-globin gene mutations that apparently contradict this rule. The corresponding ␤-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type ␤-globin (␤ WT ) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between ␤WT mRNA and ␤-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; ␤39). Functional analyses of these mRNAs with 5-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5 terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50 -54 nt boundary rule." These observations impact on current models of NMD.Nonsense-mediated mRNA decay (NMD) 1 is an mRNA surveillance mechanism that rapidly degrades mRNAs carrying premature translation termination codons (1). Nonsense-containing mRNAs targeted by NMD can be generated by naturally occurring frameshift and nonsense mutations, splicing errors, leaky 40 S scanning, and utilization of minor AUG initiation sites (2, 3). A major function of the NMD pathway is to block the synthesis of truncated proteins that could have dominant negative effects on cell function (2, 4).Recent studies have shown that the NMD pathway in mammalian cells is linked to splicing-dependent deposition of a protein complex 20 -24 nucleotides (nt) 5Ј of each exon-exon junction (exon-junction complex; EJC). The EJC contains the general splicing activator RNPS1, the RNA export factor Aly/ REF, the shuttling protein Y14, the nuclear matrix-localized serine-arginine-containing protein SRm160, the oncoprotein DEK, and the Y14 binding protein magoh. The interaction of magoh with Y14 may have a role in cytoplasmic localization of mRNAs and in anchoring the NMD-specific factors Upf3 and Upf2 to the mRNA (5-18). Previous published data have shown that Upf3 and Upf2 join the EJC in different subcellular compartments: Upf3 (Upf3a and Upf3b) is loaded onto mRNAs in the nucleus during splicing via interactions with components of the EJC. In contrast, Upf2 joins the complex soon after cytoplasmic export is initiated (14,19,20). According to the present models, translating ribosomes displace EJCs from the open reading frame (ORF) during the "pioneer" round of cytoplasmic translation (21). If, however, the mRNA contains a pr...

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 alpha-globin mRNA (h alpha-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, alpha CP. Documentation of this RNA-protein alpha-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 alpha CP is stably bound to h alpha-globin mRNA in vivo, that alpha-complex assembly on the h alpha-globin mRNA is restricted to the 3'UTR C-rich motif, and that alpha-complex assembly extends the physical half-life of h alpha-globin mRNA selectively in erythroid cells. Significantly, these studies also reveal that an artificially tethered alpha CP has the same mRNA-stabilizing activity as the native alpha-complex. These data demonstrate a unique contribution of the alpha-complex to h alpha-globin mRNA stability and support a model in which the sole function of the C-rich motif is to selectively tether alpha CP to a subset of mRNAs. Once bound, alpha CP appears to be fully sufficient to trigger downstream events in the stabilization pathway.

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 m 7 GTP cap-independent translation and identified putative IRES elements in encephalomyocarditis virus, vascular endothelial growth factor, hypoxia-inducible factors (HIFs) 1␣ and 2␣, 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 O 2 . Furthermore, although HIF-1␣ 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-1␣ and ␤-actin protein levels mirror alterations in corresponding mRNA abundance. In addition, our data suggest that cyclin-dependent kinase inhibitor p57 Kip2 and vascular endothelial growth factor mRNAs are selectively translated by an IRES-independent mechanism under hypoxic stress.Hypoxic stress is a central component of normal development and physiology as well as the pathology of multiple human diseases (1-4). Cells adapt to low oxygen (O 2 ) levels by altering their mRNA expression and translation profiles (5, 6). Most of the transcriptional effects are mediated by a family of hypoxia inducible factors (HIFs) 2 that transactivate genes by binding to hypoxia response elements in their promoters, introns, and/or enhancers (4, 7). In addition to the transcriptional effects mediated by HIF, hypoxic stress is associated with energy starvation (8, 9) and alterations in cell cycle progression (10, 11). Furthermore, whereas global protein synthesis is attenuated under low O 2 , select mRNAs believed to be involved in the adaptive response to hypoxia are preferentially translated.Hypoxia-mediated inhibition of general protein synthesis (12) is regulated primarily by the modification of eukaryotic translation initiation factors (eIFs) at two steps; that is, regeneration of the "ternary complex" (eIF2-GTP and met-tRNA) and regulation of the m 7