Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides

No abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55962/1/21079_ftp.pd

Small RNAs: Their applications and biological functions in neural development.

Recently, research on different organisms has revealed a new layer of gene regulation by small RNAs, which have sequence-specific inhibitory effects on gene expression. Long double-stranded RNAs are processed into small interfering RNAs (shRNAs), which are incorporated into the RNA-induced silencing complex and mediate target mRNA cleavage. Synthetic shRNAs also inhibit gene expression and have become effective tools for studying gene function. As an alternative to synthetic shRNAs, we designed DNA vectors expressing short hairpin RNAs (shRNAs) in cells. Designs for shRNAs were evaluated using in vitro transcription from oligonucleotide templates. An shRNA expression vector with a U6 RNA polymerase III promoter expressed shRNAs with defined ends and inhibited gene expression efficiently. We further examined the effects of length and loop sequence on inhibition by shRNAs to optimize our designs. Cotransfected shRNA vectors do not interfere with each other, and two genes can be inhibited simultaneously. We also tested shRNA vectors in dissociated cortical primary neurons and organotypic brain slices. Our results show that the use of shRNA vectors provides a versatile approach for analyzing gene function during neural development. MicroRNAs (miRNAs) are endogenous non-coding small RNAs with diverse regulatory roles. Most miRNAs interact with 3' untranslated region of the target genes through sequence complementarity and regulate the stability or translational efficiency of target mRNAs. We found that miRNA miR-124a affects neurite outgrowth during neuronal differentiation. Expression of miR-124a in differentiating P19 cells promoted neurite outgrowth, while blocking of miR-124a function delayed neurite outgrowth. In uncommitted P19 cells, miR-124a led to disruption of actin filaments and stabilization of microtubules, which resemble the cytoskeleton remodeling during neuronal morphogenesis. Rho GTPases, including Rac, Rho, and Cdc42, affect neuronal development through regulation of microfilaments and microtubules. miR-124a decreased protein levels of Cdc42 and affected subcellular localization of Rac1. Furthermore, inhibition of Rac1 activities in differentiating P19 cells promoted neurite outgrowth; and activation of either Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124a. These results suggest that Cdc42 and Rac1 may function downstream of miR-124a in promoting neurite outgrowth. Altered neurite outgrowth was also observed in mouse primary cortical neurons when the expression level of miR-124a was increased, or when miR-124a function was blocked. These results indicate that miRNAs can contribute to the regulation of neurite outgrowth in neuronal differentiation.Ph.D.Biological SciencesNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125786/2/3208587.pd

Candidate Modifier Genes for the Penetrance of Leber’s Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a maternally transmitted disease caused by mitochondria DNA (mtDNA) mutation. It is characterized by acute and subacute visual loss predominantly affecting young men. The mtDNA mutation is transmitted to all maternal lineages. However, only approximately 50% of men and 10% of women harboring a pathogenic mtDNA mutation develop optic neuropathy, reflecting both the incomplete penetrance and its unexplained male prevalence, where over 80% of patients are male. Nuclear modifier genes have been presumed to affect the penetrance of LHON. With conventional genetic methods, prior studies have failed to solve the underlying pathogenesis. Whole exome sequencing (WES) is a new molecular technique for sequencing the protein-coding region of all genes in a whole genome. We performed WES from five families with 17 members. These samples were divided into the proband group (probands with acute onset of LHON, n = 7) and control group (carriers including mother and relative carriers with mtDNSA 11778 mutation, without clinical manifestation of LHON, n = 10). Through whole exome analysis, we found that many mitochondria related (MT-related) nuclear genes have high percentage of variants in either the proband group or control group. The MT genes with a difference over 0.3 of mutation percentage between the proband and control groups include AK4, NSUN4, RDH13, COQ3, and FAHD1. In addition, the pathway analysis revealed that these genes were associated with cofactor metabolism pathways. Family-based analysis showed that several candidate MT genes including METAP1D (c.41G > T), ACACB (c.1029del), ME3 (c.972G > C), NIPSNAP3B (c.280G > C, c.476C > G), and NSUN4 (c.4A > G) were involved in the penetrance of LHON. A GWAS (genome wide association study) was performed, which found that ADGRG5 (Chr16:575620A:G), POLE4 (Chr2:7495872T:G), ERMAP (Chr1:4283044A:G), PIGR (Chr1:2069357C:T;2069358G:A), CDC42BPB (Chr14:102949A:G), PROK1 (Chr1:1104562A:G), BCAN (Chr 1:1566582C:T), and NES (Chr1:1566698A:G,1566705T:C, 1566707T:C) may be involved. The incomplete penetrance and male prevalence are still the major unexplained issues in LHON. Through whole exome analysis, we found several MT genes with a high percentage of variants were involved in a family-based analysis. Pathway analysis suggested a difference in the mutation burden of MT genes underlining the biosynthesis and metabolism pathways. In addition, the GWAS analysis also revealed several candidate nuclear modifier genes. The new technology of WES contributes to provide a highly efficient candidate gene screening function in molecular genetics

Akt Regulates Basic Helix-Loop-Helix Transcription Factor-Coactivator Complex Formation and Activity during Neuronal Differentiation

Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation