miR-328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts.

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

Multi-omic profiling of pituitary thyrotropic cells and progenitors.

BACKGROUND: The pituitary gland is a neuroendocrine organ containing diverse cell types specialized in secreting hormones that regulate physiology. Pituitary thyrotropes produce thyroid-stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate, but the transcriptomic and epigenomic landscapes of these neuroendocrine cells have not been characterized. The goal of this work was to discover transcriptional regulatory elements that drive thyrotrope fate. RESULTS: We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an undifferentiated Pou1f1 lineage progenitor (GHF-T1) and a committed thyrotrope line that produces TSH (TαT1). We compared RNA-seq, ATAC-seq, histone modification (H3K27Ac, H3K4Me1, and H3K27Me3), and POU1F1 binding in these cell lines. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding at unique sites. We validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element. CONCLUSION: These results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis.

A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript.

The establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

PolyC-binding proteins enhance expression of the CDK2 cell cycle regulatory protein via alternative splicing.

The PolyC binding proteins (PCBPs) impact alternative splicing of a subset of mammalian genes that are enriched in basic cellular functions. Here, we focus our analysis on PCBP-controlled cassette exon-splicing within the cell cycle control regulator cyclin-dependent kinase-2 (CDK2) transcript. We demonstrate that PCBP binding to a C-rich polypyrimidine tract (PPT) preceding exon 5 of the CDK2 transcript enhances cassette exon inclusion. This splice enhancement is U2AF65-independent and predominantly reflects actions of the PCBP1 isoform. Remarkably, PCBPs' control of CDK2 ex5 splicing has evolved subsequent to mammalian divergence via conversion of constitutive exon 5 inclusion in the mouse CDK2 transcript to PCBP-responsive exon 5 alternative splicing in humans. Importantly, exclusion of exon 5 from the hCDK2 transcript dramatically represses the expression of CDK2 protein with a corresponding perturbation in cell cycle kinetics. These data highlight a recently evolved post-transcriptional pathway in primate species with the potential to modulate cell cycle control.

Functional characterization of a human POU1F1 mutation associated with isolated growth hormone deficiency: a novel etiology for IGHD.

POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHß transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.

Cytoplasmic poly(A) binding protein-1 binds to genomically encoded sequences within mammalian mRNAs.

The functions of the major mammalian cytoplasmic poly(A) binding protein, PABPC1, have been characterized predominantly in the context of its binding to the 3' poly(A) tails of mRNAs. These interactions play important roles in post-transcriptional gene regulation by enhancing translation and mRNA stability. Here, we performed transcriptome-wide CLIP-seq analysis to identify additional PABPC1 binding sites within genomically encoded mRNA sequences that may impact on gene regulation. From this analysis, we found that PABPC1 binds directly to the canonical polyadenylation signal in thousands of mRNAs in the mouse transcriptome. PABPC1 binding also maps to translation initiation and termination sites bracketing open reading frames, exemplified most dramatically in replication-dependent histone mRNAs. Additionally, a more restricted subset of PABPC1 interaction sites comprised A-rich sequences within the 5' UTRs of mRNAs, including Pabpc1 mRNA itself. Functional analyses revealed that these PABPC1 interactions in the 5' UTR mediate both auto- and trans-regulatory translational control. In total, these findings reveal a repertoire of PABPC1 binding that is substantially broader than previously recognized with a corresponding potential to impact and coordinate post-transcriptional controls critical to a broad array of cellular functions.

Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster.

The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR 'loops' over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed 'hCS chromatin hub'. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster.

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.

Autonomous actions of the human growth hormone long-range enhancer.

The human growth hormone (hGH) gene is controlled by a long-range enhancer, HSI, located 14.5 kb 5' to the hGH promoter. HSI establishes a domain of noncoding transcription that is 'looped' to the hGH promoter as an essential step in initiating hGH gene expression. Thus, defining how HSI generates its domain of noncoding transcription is central to understanding its long-range function. Here, we demonstrate that activation of noncoding transcription reflects an HSI-autonomous activity fully independent of interactions with linked gene promoters and occurring in spatial and temporal synchrony with initiation of GH expression in the embryonic pituitary. HSI establishes its noncoding transcription start sites (TSS) over a defined distance from its core determinants and in a manner independent of local primary sequences. The interval between HSI and it TSS co-maps with a domain of disordered and/or highly mobile nucleosomes specific to the pituitary locus. Thus, a localized chromatin reconfiguration by HSI and consequent establishment of an adjacent domain of noncoding transcription constitute initiating events in long-range enhancer function within the hGH locus.

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human ß-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the 'AUG-proximity effect'. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3' to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.

Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia.

Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context-specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3' enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.

Tissue specific CTCF occupancy and boundary function at the human growth hormone locus.

The robust and tissue-specific activation of the human growth hormone (hGH) gene cluster in the pituitary and placenta constitutes an informative model for analysis of gene regulation. The five-gene hGH cluster is regulated by two partially overlapping sets of DNase I hypersensitive sites (HSs) that constitute the pituitary (HSI, II, III and V) and placental (HSIII, IV, and V) locus control regions (LCRs). The single placenta-specific LCR component, HSIV, is located at -30 kb to the cluster. Here we generate a series of hGH/BAC transgenes specifically modified to identify structural features of the hGH locus required for its appropriate placental expression. We find that placental specificity is dependent on the overall multigene configuration of the cluster whereas the distance between the cluster and its LCR impacts the level of placental expression. We further observe that a major function of the placental hGH LCR is to insulate the transgene locus from site-of-integration effects. This insulation activity is linked to placenta-specific occupancy of the chromatin architectural protein, CTCF, at HSIV. These data reveal a remarkable combination of structural configurations and regulatory determinants that must work in concert to insure robust and tightly controlled expression from a complex multigene locus.

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.

Identification of a secondary promoter within the human B cell receptor component gene hCD79b.

The human B cell-specific protein, CD79b (also known as Igß and B29) constitutes an essential signal transduction component of the B cell receptor. Although its function is central to the triggering of B cell terminal differentiation in response to antigen stimulation, the transcriptional determinants that control CD79b gene expression remain poorly defined. In the present study, we explored these determinants using a series of hCD79b transgenic mouse models. Remarkably, we observed that the previously described hCD79b promoter along with its associated enhancer elements and first exon could be deleted without appreciable loss of hCD79b transcriptional activity or tissue specificity. In this deletion setting, a secondary promoter located within exon 2 maintained full levels and specificity of hCD79b transcription. Of note, this secondary promoter was also active, albeit at lower levels, in the wild-type hCD79b locus. The activity of the secondary promoter was dependent on the action(s) of a conserved sequence element mapping to a chromatin DNase I hypersensitive site located within intron 1. mRNA generated from this secondary promoter is predicted to encode an Igß protein lacking a signal sequence and thus unable to serve normal B cell receptor function. Although the physiologic role of the hCD79b secondary promoter and its encoded protein remain unclear, the current data suggest that it has the capacity to play a role in normal as well as pathologic states in B cell proliferation and function.

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

DNase I hypersensitive site II of the human growth hormone locus control region mediates an essential and distinct long-range enhancer function.

Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

Haploinsufficiency of the autism candidate gene Neurobeachin induces autism-like behaviors and affects cellular and molecular processes of synaptic plasticity in mice.

Neurobeachin (NBEA), a brain-enriched multidomain scaffolding protein involved in neurotransmitter release and synaptic functioning, has been identified as a candidate gene for autism spectrum disorder (ASD) in four unrelated patients haploinsufficient for NBEA. The aim of this study was to map the behavioral phenotype of Nbea(+/-) mice in order to understand its contribution to the pathogenesis of ASD. ASD-like behavioral variables of Nbea(+/-) mice were related to basal neuronal activity in different brain regions by in situ hybridizations and extracellular field recordings of synaptic plasticity in hippocampal cornu ammonis 1 (CA1) region. Levels of BDNF and phosphorylated cAMP response element-binding protein (CREB) were measured in an attempt to investigate putatively underlying changes in these neuromolecules. Nbea(+/-) mice exhibit several ASD-like features, including changes in self-grooming behavior, social behaviors, conditioned fear responses, and spatial learning and memory, which coincided with enhanced long-term potentiation (LTP) in their CA1 region. The observed alterations in learning and memory and hippocampal LTP are concomitant with decreased expression of the immediate early gene zif268 in dorsomedial striatum and hippocampal CA1 region, increased CREB phosphorylation, and increased hippocampal BDNF expression. These findings indicate that Nbea haploinsufficiency leads to various molecular and cellular changes that affect neuroplasticity and behavioral functions in mice, and could thus underlie the ASD symptomatology in NBEA deficient humans.

The poly(rC)-binding protein alphaCP2 is a noncanonical factor in X. laevis cytoplasmic polyadenylation.

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 3' UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein αCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by αCP2. We find that the hα-globin 3' UTR, a validated mammalian αCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich αCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XαCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich αCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XαCP2 and CPEB1. These studies establish XαCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings.

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 kb 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.

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.