microRNA-mediated Gene Regulation Research Articles

MicroRNA-Mediated Gene Regulation Effectively Restricts In Vivo Transgene Expression in Hematopoietic Stem Cell Progeny.

Stem cell engineering and targeted in vivo gene delivery increasingly require tight control of transgene expression. Lineage- and differentiation stage-specific gene regulation is classically afforded by pol-II-dependent transcript regulation. A super-imposed layer of post-transcriptional control would be valuable to correct undesirable expression patterns or fine-tune developmentally regulated or inducible gene expression. microRNAs (miRNAs) have recently emerged as potent repressors of gene expression at the post-transcriptional level. In this study, we investigate the potential of using miRNA regulation to provide lineage-restricted expression, exploiting miRNAs with distinctive expression patterns in hematopoietic tissue. miR-223 is preferentially expressed in granulocytes and monocytes (80-and 110-fold, respectively). miR-181a is highly expressed in B-lymphocytes and, particularly, in thymocytes, but down-regulated (∼1000-fold) in post-thymic T cells. We, therefore, constructed lentiviral vectors encoding either green fluorescent protein (GFP) or an antigen-specific receptor, placed under the transcriptional control of the ubiquitous EF1a promoter and tagged with miRNA-recognition elements (MREs) complementary to the mature miR-223 or miR-181a. In a panel of murine and human cell lines expressing varying levels of the two miRNAs we find that GFP knockdown is dependent on the presence of the miRNA and directly proportional to the number of MRE repeats. Four copies of the repeat permit better down-regulation than 2 copies (albeit depending on the level of endogenous miRNA). Two different MREs can be combined in tandem, resulting in additive down-regulation. In vivo, in mouse bone marrow chimeras harboring the miR-223-responsive vector, GFP expression is specifically repressed in myeloid cells (>85% compared to the control vector lacking miRNA target sequences). Reciprocally, chimeras harboring the miR-181a-regulated vector express GFP in myeloid and erythroid lineages, but transgene expression is profoundly repressed in thymocytes and B-cell progenitors. A vector harboring a composite MRE confers GFP expression almost exclusively confined to the erythroid lineage. These results demonstrate for the first time that transgene expression can be selectively regulated at the post-transcriptional level within the hematopoietic tissue. In mouse chimeras expressing a CD19-specific antigen receptor (which we and others are currently using in clinical trials in B cell malignancies), we analyze receptor expression in the 4 double negative subsets (DN1, DN2, DN3 and DN4), the double-positive subset (DP), and the CD4+ and CD8+ single positive (SP) subsets. We detail and quantify receptor knock-down at each developmental stage, and show that miR-181a-mediated regulation prevents receptor expression at critical stages of positive and negative thymic selection. Antigen receptor expression is dramatically repressed in DN and DP cells, while rising in CD4+ and CD8+ SP thymocytes. Importantly, expression is fully restored in post-thymic T cells, and maintained in activated T cells. miRNA-mediated post-transcriptional regulation is thus proving to be a powerful means to direct lineage- and differentiation stage-specific transgene expression in genetically modified stem cells.

Clinical and neuropathologic study of a French family with a mutation in the neuroserpin gene

Familial encephalopathy with neuroserpin inclusion bodies is a recently described neurodegenerative disease that is responsible for progressive myoclonic epilepsy or presenile dementia. In a French family with the S52R mutation of the neuroserpin gene, progressive myoclonic epilepsy was associated with a frontal syndrome. The typical cerebral inclusions (Collins bodies) were abundant in the frontal cortex and in the head of the caudate nucleus but spared the cerebellum.

MIWI2 Is Essential for Spermatogenesis and Repression of Transposons in the Mouse Male Germline

Small RNAs associate with Argonaute proteins and serve as sequence-specific guides for regulation of mRNA stability, productive translation, chromatin organization, and genome structure. In animals, the Argonaute superfamily segregates into two clades. The Argonaute clade acts in RNAi and in microRNA-mediated gene regulation in partnership with 21-22 nt RNAs. The Piwi clade, and their 26-30 nt piRNA partners, have yet to be assigned definitive functions. In mice, two Piwi-family members have been demonstrated to have essential roles in spermatogenesis. Here, we examine the effects of disrupting the gene encoding the third family member, MIWI2. Miwi2-deficient mice display a meiotic-progression defect in early prophase of meiosis I and a marked and progressive loss of germ cells with age. These phenotypes may be linked to an inappropriate activation of transposable elements detected in Miwi2 mutants. Our observations suggest a conserved function for Piwi-clade proteins in the control of transposons in the germline.

Principles and effects of microRNA-mediated post-transcriptional gene regulation

MicroRNAs (miRNAs) are abundant regulatory RNAs involved in the regulation of many key biological processes. Recent advances in understanding the mechanism of RNA interference and miRNA-mediated mechanisms shed light on major principals of the formation of the regulatory complex and provide models to explain how these small regulatory RNA species interfere with gene expression and how they influence the translational status of the transcriptome.

A germline-specific class of small RNAs binds mammalian Piwi proteins

Small RNAs associate with Argonaute proteins and serve as sequence-specific guides to regulate messenger RNA stability, protein synthesis, chromatin organization and genome structure. In animals, Argonaute proteins segregate into two subfamilies. The Argonaute subfamily acts in RNA interference and in microRNA-mediated gene regulation using 21-22-nucleotide RNAs as guides. The Piwi subfamily is involved in germline-specific events such as germline stem cell maintenance and meiosis. However, neither the biochemical function of Piwi proteins nor the nature of their small RNA guides is known. Here we show that MIWI, a murine Piwi protein, binds a previously uncharacterized class of approximately 29-30-nucleotide RNAs that are highly abundant in testes. We have therefore named these Piwi-interacting RNAs (piRNAs). piRNAs show distinctive localization patterns in the genome, being predominantly grouped into 20-90-kilobase clusters, wherein long stretches of small RNAs are derived from only one strand. Similar piRNAs are also found in human and rat, with major clusters occurring in syntenic locations. Although their function must still be resolved, the abundance of piRNAs in germline cells and the male sterility of Miwi mutants suggest a role in gametogenesis.

MicroRNAs as Oncogenes and Tumor Suppressors

Recent discoveries related to microRNAs, RNA interference, small interfering RNAs (siRNAs), and small modulatory RNAs have revealed a new class of mechanisms of gene regulation that are mediated by small, noncoding RNAs. Among these small RNAs are the microRNAs. These are thought to control gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs (mRNAs). Surprisingly simple and elegant, microRNA-mediated gene regulation is guided by the base-pairing rules of Watson and Crick.MicroRNAs are individually encoded by their own set of genes and are an integral component of the genetic program. Some are located in noncoding regions . . .

The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation.

The Arabidopsis HYL1 gene encodes a nuclear double-stranded RNA-binding protein. A knockout mutation of the hyl1 gene is recessive and pleiotropic, causing developmental abnormalities, increasing sensitivity to abscisic acid, and reducing sensitivity to auxin and cytokinin. We report that levels of several microRNAs (miRNAs; miR159, -167, and -171) are reduced in homozygous mutant plants, and levels of two of three tested target mRNAs are elevated. Conversely, the miRNA levels are elevated in plants expressing a HYL1 cDNA from a strong promoter, and the corresponding target RNAs are reduced. These changes result from alterations in the stability of the target RNAs. However, double-stranded RNA-induced posttranscriptional gene silencing is unaffected by the hyl1 mutation. One-third to one-half of the cellular HYL1 protein is in a macromolecular complex, and a GFP-HYL1 fusion protein is found predominantly in the nucleus, although it is observed in both nucleus and cytoplasm in some cells. Within nuclei, HYL1 is associated with subnuclear bodies and ring-like structures. These observations provide evidence that the HYL1 protein is part of a nuclear macromolecular complex that is involved in miRNA-mediated gene regulation. Because hyl1 mutants show marked abnormalities in hormone responses, these results further suggest that miRNA-mediated changes in mRNA stability play a vital role in plant hormone signaling.