The RNaseIII-containing enzyme Dicer is believed to be required for the processing of most, if not all, microRNAs (miRNAs) and for processing long dsRNA into small interfering RNAs. Because the complete loss of Dicer in both zebrafish and mice results in early embryonic lethality, it has been impossible to determine what role, if any, Dicer has in patterning later tissues in the developing vertebrate embryo. To bypass the early requirement of Dicer in development, we have created a conditional allele of this gene in mice. Using transgenes to drive Cre expression in discrete regions of the limb mesoderm, we find that removal of Dicer results in the loss of processed miRNAs. Phenotypically, developmental delays, in part due to massive cell death as well as disregulation of specific gene expression, lead to the formation of a much smaller limb. Thus, Dicer is required for the formation of normal mouse limbs. Strikingly, however, we did not detect defects in basic patterning or in tissue-specific differentiation of Dicer-deficient limb buds.microRNAs ͉ mouse
Animal development is an extremely robust process resulting in stereotyped outcomes. Canalization is a design principle wherein developmental pathways are stabilized to increase phenotypic reproducibility. Recent revelations into microRNA (miRNA) function suggest that miRNAs act as key players in canalizing genetic programs. We suggest that miRNA interactions with the network of protein-coding genes evolved to buffer stochastic perturbations and thereby confer robustness to developmental genetic programs.
To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.
Vertebrate mRNAs are frequently targeted for post-transcriptional repression by microRNAs (miRNAs) through mechanisms involving pairing of 39 UTR seed matches to bases at the 59 end of miRNAs. Through analysis of expression array data following miRNA or siRNA overexpression or inhibition, we found that mRNA fold change increases multiplicatively (i.e., log-additively) with seed match count and that a single 8 mer seed match mediates down-regulation comparable to two 7 mer seed matches. We identified several targeting determinants that enhance seed match-associated mRNA repression, including the presence of adenosine opposite miRNA base 1 and of adenosine or uridine opposite miRNA base 9, independent of complementarity to the siRNA/miRNA. Increased sequence conservation in the ;50 bases 59 and 39 of the seed match and increased AU content 39 of the seed match were each independently associated with increased mRNA down-regulation. All of these determinants are enriched in the vicinity of conserved miRNA seed matches, supporting their activity in endogenous miRNA targeting. Together, our results enable improved siRNA off-target prediction, allow integrated ranking of conserved and nonconserved miRNA targets, and show that targeting by endogenous and exogenous miRNAs/siRNAs involves similar or identical determinants.
Differentiated cell types derived from human embryonic stem cells (hESCs) may serve in the future to treat various human diseases. A crucial step toward their successful clinical application is to examine the immune response that might be launched against them after transplantation. We used two experimental platforms to examine the in vivo leukocyte response toward hESCs. First, immunocompetent and immunodeficient mouse strains were used to identify T cells as the major component that causes xenorejection of hESCs. Second, mice that were conditioned to carry peripheral blood leukocytes from human origin were used to test the human leukocyte alloresponse toward undifferentiated and differentiated hESCs. Using this model, we have detected only a minute immune response toward undifferentiated as well as differentiated hESCs over the course of 1 month, although control adult grafts were repeatedly infiltrated with lymphocytes and destroyed. Our data show that the cells evade immune destruction due to a low immunostimulatory potential. Nevertheless, a human cytotoxic T lymphocyte clone that was specifically prepared to recognize two hESC lines could lyse the cells after major histocompatibility complex class I (MHC-I) induction. Although MHC-I levels in hESCs are sufficient for rejection by cytotoxic T cells, our data suggest that the immunostimulatory capacity of the cells is very low. Thus, immunosuppressive regimens for hESC-based therapeutics could be highly reduced compared with conventional organ transplantation because direct allorejection processes of hESCs and their derivatives are considerably weaker. STEM CELLS 2006;24:221-229
Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1–3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.
MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.
Defective RNA metabolism is an emerging mechanism involved in ALS pathogenesis and possibly in other neurodegenerative disorders. Here, we show that microRNA (miRNA) activity is essential for long-term survival of postmitotic spinal motor neurons (SMNs) in vivo. Thus, mice that do not process miRNA in SMNs exhibit hallmarks of spinal muscular atrophy (SMA), including sclerosis of the spinal cord ventral horns, aberrant end plate architecture, and myofiber atrophy with signs of denervation. Furthermore, a neurofilament heavy subunit previously implicated in motor neuron degeneration is specifically up-regulated in miRNA-deficient SMNs. We demonstrate that the heavy neurofilament subunit is a target of miR-9, a miRNA that is specifically down-regulated in a genetic model of SMA. These data provide evidence for miRNA function in SMN diseases and emphasize the potential role of miR-9-based regulatory mechanisms in adult neurons and neurodegenerative states.ALS | Dicer | microRNA | motor neuron | neurodegeneration
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