Yingqun Huang

The Imprinted H19 LncRNA Antagonizes Let-7 MicroRNAs

Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs.

SR Splicing Factors Serve as Adapter Proteins for TAP-Dependent mRNA Export.

The only mammalian RNA binding adapter proteins known to partner with TAP/NXF1, the primary receptor for general mRNA export, are members of the REF family. We demonstrate that at least three shuttling SR (serine/arginine-rich) proteins interact with the same domain of TAP/NXF1 that binds REFs. Included are 9G8 and SRp20, previously shown to promote the export of intronless RNAs. A peptide derived from the N terminus of 9G8 inhibits the binding of both REF and SR proteins to TAP/NXF1 in vitro, and this finding argues for competitive interactions. In Xenopus oocytes, the N terminus of 9G8 exhibits a dominant-negative effect on mRNA export from the nucleus, while addition of excess TAP/NXF1 overcomes this inhibition. Thus, multiple adapters including SR proteins most likely cooperate to recruit multiple copies of TAP/NXF1 for efficient mRNA export.

Splicing Factors SRp20 and 9G8 Promote the Nucleocytoplasmic Export of mRNA

We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export.

Pluripotency factors Lin28 and Oct4 identify a sub-population of stem cell-like cells in ovarian cancer

Lin28 and Oct4 are highly expressed in human embryonic stem (ES) cells and, along with two other stem cell marker proteins (Nanog and Sox2), together can convert human somatic cells to pluripotency. As an RNA-binding protein, Lin28 acts to stimulate the translation of a specific subset of mRNAs, and to inhibit the biogenesis of a group of microRNAs. Oct4 is a transcription factor essential for the maintenance of pluripotency and survival of ES cells. In this study, we report that a sub-population of epithelial ovarian cancer (EOC) cells co-expresses Lin28 and Oct4 as demonstrated in the analyses of both cell lines and patient tumor samples. We also observe that the combined expression of these proteins in tumor samples is correlated with advanced tumor grade. Intriguingly, when the expression of these two proteins is repressed in the same cells using RNA interference, there is significant reduction in cell growth and survival. We thus propose that Lin28 and Oct4 may have important roles in the initiation and/or progression of EOC, and consequently may serve as important molecular diagnostics and/or therapeutic targets for the development of novel treatment strategies in EOC patients.

Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells

Lin28 acts as a repressor of microRNA processing and as a post-transcriptional regulatory factor for a subset of mRNAs. Here we report that in human embryonic stem cells Lin28 facilitates the expression of the pivotal pluripotency factor Oct4 at the post-transcriptional level. We provide evidence that Lin28 binds Oct4 mRNA directly through high affinity sites within its coding region and that an interaction between Lin28 and RNA helicase A (RHA) may play a part in the observed regulation. We further demonstrate that decreasing RHA levels impairs Lin28-dependent stimulation of translation in a reporter system. Taken together with previous studies showing that RHA is required for efficient translation of a specific class of mRNAs, these findings suggest a novel mechanism by which Lin28 may affect target mRNA expression and represent the first evidence of post-transcriptional regulation of Oct4 expression by Lin28 in human embryonic stem cells.

Genome‐Wide Studies Reveal That Lin28 Enhances the Translation of Genes Important for Growth and Survival of Human Embryonic Stem Cells

Lin28 inhibits the expression of let‐7 microRNAs but also exhibits let‐7‐independent functions. Using immunoprecipitation and deep sequencing, we show here that Lin28 preferentially associates with a small subset of cellular mRNAs. Of particular interest are those for ribosomal proteins and metabolic enzymes, the expression levels of which are known to be coupled to cell growth and survival. Polysome profiling and reporter analyses suggest that Lin28 stimulates the translation of many or most of these targets. Moreover, Lin28‐responsive elements were found within the coding regions of all target genes tested. Finally, a mutant Lin28 that still binds RNA but fails to interact with RNA helicase A (RHA), acts as a dominant‐negative inhibitor of Lin28‐dependent stimulation of translation. We suggest that Lin28, working in concert with RHA, enhances the translation of genes important for the growth and survival of human embryonic stem cells. STEM CELLS 2011;496–504

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.

Regulation of tumor cell migration and invasion by the H19/let-7 axis is antagonized by metformin-induced DNA methylation.

The imprinted, developmentally regulated H19 long noncoding RNA has been implicated in the pathogenesis of diverse human cancers, but the underlying mechanisms have remained poorly understood. Here, we report that H19 promotes tumor cell migration and invasion by inhibiting let-7, a potent tumor suppressor microRNA that functions to posttranscriptionally suppress the expression of oncogenes that regulate cell growth and motility. We show that H19 depletion impairs, whereas its overexpression enhances the motility and invasiveness of tumor cells. These phenomena occur, at least in part through affecting let-7-mediated regulation of metastasis-promoting genes, including Hmga2, c-Myc and Igf2bp3. This H19/let-7-dependent regulation is recapitulated in vivo where co-expressions of oncogenes and H19 exist in both primary human ovarian and endometrial cancers. Furthermore, we provide evidence that the anti-diabetic drug metformin inhibits tumor cell migration and invasion, partly by downregulating H19 via DNA methylation. Our results reveal a novel mechanism underpinning H19-mediated regulation in metastasis and may explain why in some cases increased let-7 expression unexpectedly correlates with poor prognosis, given the widely accepted role for let-7 as a tumor suppressor. Targeting this newly identified pathway might offer therapeutic opportunities.

Fragile X mental retardation protein FMRP and the RNA export factor NXF2 associate with and destabilize Nxf1 mRNA in neuronal cells

Fragile X syndrome is caused by the inactivation of the X-linked FMR1 gene, leading to the loss of its encoded protein FMRP. Although macroorchidism and defects in neuronal architecture and function have been associated with lack of FMRP, the exact molecular mechanism underlying this disease remains unclear. We have reported previously that in the brain and testis of mice, FMRP specifically interacts with a distinct mRNA nuclear export factor NXF2 but not with its close relative NXF1, a ubiquitously expressed essential mRNA nuclear export factor. This interaction marked NXF2 as a putative functional partner of FMRP. Here, we demonstrate by immunoprecipitation and quantitative real-time RT-PCR that, in cultured mouse neuronal cells, both FMRP and NXF2 are present in Nxf1 mRNA-containing ribonucleoprotein particles. Further, we show that expression of NXF2 leads to the destabilization of Nxf1 mRNA and that this effect is abolished when Fmr1 expression is reduced by siRNA, arguing that both proteins collaborate to exert this effect. Importantly, these findings correlate well with our observations that in both mouse hippocampal neurons and male germ cells where the expression of FMRP and NXF2 is most prominent, the expression of NXF1 is relatively poorly expressed. Our studies thus identify Nxf1 mRNA as a likely biologically relevant in vivo target of both FMRP and NXF2 and implicate FMRP, in conjunction with NXF2, as a posttranscriptional regulator of a major mRNA export factor. Such regulation may prove important in the normal development and function of neurons as well as of male germ cells.

A mirror of two faces: Lin28 as a master regulator of both miRNA and mRNA.

Lin28 is an evolutionarily conserved RNA‐binding protein that plays important roles in development, pluripotency, tumorigenesis, and metabolism. Emerging evidence suggests that the pleiotropic roles of Lin28 in the diverse physiological and pathological processes are mechanistically linked to its ability to modulate not only the biogenesis of miRNAs, particularly the let‐7 family miRNAs, but also the translation of mRNAs important for cell growth and metabolism. Let‐7 negatively regulates the translation of oncogenes, cell cycle regulators, and metabolic pathway components. Lin28 relieves this repression by blocking the production of mature let‐7. Lin28 binds to the terminal loops of let‐7 precursors, leading to inhibition of processing and the induction of uridylation and precursor degradation. Lin28 also is a direct translational regulator: it selectively binds to a cohort of mRNAs and stimulates their translation. Recent advances in our understanding of Lin28‐mediated mechanisms of posttranscriptional regulation of gene expression reveal important roles of this protein in the fields of development, stem cells, metabolic diseases, and cancer. WIREs RNA 2012, 3:483–494. doi: 10.1002/wrna.1112