Target mRNA Molecules Research Articles

MicroRNAs and Neurodegenerative Diseases.

microRNAs (miRNAs) are small, noncoding RNA molecules that through imperfect base-pairing with complementary sequences of target mRNA molecules, typically cleave target mRNA, causing subsequent degradation or translation inhibition. Although an increasing number of studies have identified misregulated miRNAs in the neurodegenerative diseases (NDDs) Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, which suggests that alterations in the miRNA regulatory pathway could contribute to disease pathogenesis, the molecular mechanisms underlying the pathological implications of misregulated miRNA expression and the regulation of the key genes involved in NDDs remain largely unknown. In this chapter, we provide evidence of the function and regulation of miRNAs and their association with the neurological events in NDDs. This will help improve our understanding of how miRNAs govern the biological functions of key pathogenic genes in these diseases, which potentially regulate several pathways involved in the progression of neurodegeneration. Additionally, given the growing interest in the therapeutic potential of miRNAs, we discuss current clinical challenges to developing miRNA-based therapeutics for NDDs.

225 VALIDATION OF REFERENCE microRNAs FOR NORMALIZING EXPRESSION DATA GENERATED BY QUANTITATIVE PCR

Small noncoding RNAs such as microRNAs (miRNA) act as posttranscriptional regulators of numerous gene targets. The level of expression of these epigenetic factors can regulate the stability and translation of target mRNA molecules. Quantification of miRNA expression levels can therefore help us understand biological processes such as those regulating oocyte and pre-implantation embryo development. To date, most studies that have examined miRNA expression levels have used expression of either reference mRNAs or U6 spliceosomal RNA or miRNA Let-7a for normalization. We analysed the suitability of several miRNAs as potential expression normalizers in bovine oocytes and early embryos. Bovine oocytes at the germinal vesicle and metaphase II stages, and zygotes, 2-cell, 4-cell and 8-cell embryos, morulae, and blastocysts were collected during three in vitro fertilization replicates. qPCR was performed to quantify expression of miR-93, miR-103a, miR-26a, miR-191, miR-23b, Let-7a, and U6. The average starting material for each sample was determined with the help of specific standard curves made for each primer set. Subsequently, geNorm software was used to identify a set of stably transcribed miRNAs. Surprisingly, U6 spliceosomal RNA and Let-7a, which are widely used to normalise miRNA expression levels, were not stably expressed and were therefore considered unsuitable for normalization. Stepwise removal to determine the optimal number of reference miRNAs identified miR-93 and miR-103a as the most stably expressed. It is concluded that the combination of miR-93 and miR-103a can be used for normalizing miRNA expression for qPCR experiments on bovine oocytes and pre-implantation embryos.

Deciphering the function and regulation of microRNAs in Alzheimer's disease and Parkinson's disease.

MicroRNAs (miRNAs) are single stranded, noncoding RNA molecules that are encoded by eukaryotic nuclear DNA. miRNAs function through imperfect base-pairing with complementary sequences of target mRNA molecules, which is typically via the cleavage of target mRNA with transcriptional repression or translational degradation. An increasing number of studies identified dysregulation of miRNAs in neurodegenerative disease and suggest that alterations in the miRNA regulatory pathway could contribute to the disease pathogenesis. However, molecular mechanisms underlying the pathological implications of dysregulated miRNA expression and regulation of the key genes that are involved in neurodegenerative diseases remain largely unknown. Here, we review the evidence for the functional role of dysregulated miRNAs involved in disease pathogenesis, as well as how miRNAs govern neuronal functions either upstream or downstream of target genes that are disease pathogenic factors. Furthermore, we review the cellular feedback regulation between miRNAs and target genes in neurodegenerative diseases, with a focus on Alzheimer's disease and Parkinson's disease.

Fusion FISH Imaging: Single-Molecule Detection of Gene Fusion Transcripts In Situ

Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional recombinant genes, which have been implicated as the cause of a number of lymphomas, leukemias, sarcomas, and solid tumors. Reliable methods are needed for the in situ detection of the transcripts encoded by these recombinant genes. We have developed just such a method, utilizing single-molecule fluorescence in situ hybridization (sm-FISH), in which approximately 50 short fluorescent probes bind to adjacent sites on the same mRNA molecule, rendering each target mRNA molecule visible as a diffraction-limited spot in a fluorescence microscope. Utilizing this method, gene fusion transcripts are detected with two differently colored probe sets, each specific for one of the two recombinant segments of a target mRNA; enabling the fusion transcripts to be seen in the microscope as distinct spots that fluoresce in both colors. We demonstrate this method by detecting the BCR-ABL fusion transcripts that occur in chronic myeloid leukemia cells, and by detecting the EWSR1-FLI1 fusion transcripts that occur in Ewing's sarcoma cells. This technology should pave the way for accurate in situ typing of many cancers that are associated with, or caused by, fusion transcripts.

A Genotoxic Stress-Responsive miRNA, miR-574-3p, Delays Cell Growth by Suppressing the Enhancer of Rudimentary Homolog Gene in Vitro

MicroRNA (miRNA) is a type of non-coding RNA that regulates the expression of its target genes by interacting with the complementary sequence of the target mRNA molecules. Recent evidence has shown that genotoxic stress induces miRNA expression, but the target genes involved and role in cellular responses remain unclear. We examined the role of miRNA in the cellular response to X-ray irradiation by studying the expression profiles of radio-responsive miRNAs and their target genes in cultured human cell lines. We found that expression of miR-574-3p was induced in the lung cancer cell line A549 by X-ray irradiation. Overexpression of miR-574-3p caused delayed growth in A549 cells. A predicted target site was detected in the 3′-untranslated region of the enhancer of the rudimentary homolog (ERH) gene, and transfected cells showed an interaction between the luciferase reporter containing the target sequences and miR-574-3p. Overexpression of miR-574-3p suppressed ERH protein production and delayed cell growth. This delay was confirmed by knockdown of ERH expression. Our study suggests that miR-574-3p may contribute to the regulation of the cell cycle in response to X-ray irradiation via suppression of ERH protein production.

MicroRNAs: promising new antiangiogenic targets in cancer.

MicroRNAs are one class of small, endogenous, non-coding RNAs that are approximately 22 nucleotides in length; they are very numerous, have been phylogenetically conserved, and involved in biological processes such as development, differentiation, cell proliferation, and apoptosis. MicroRNAs contribute to modulating the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules and so they play a key role in both health and disease. Angiogenesis is the process of new blood vessel formation from preexisting ones, which is particularly relevant to cancer and its progression. Over the last few years, microRNAs have emerged as critical regulators of signalling pathways in multiple cell types including endothelial and perivascular cells. This review summarises the role of miRNAs in tumour angiogenesis and their potential implications as therapeutic targets in cancer.

In Vivo RNAi-Based Screens: Studies in Model Organisms

RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

Toxicity profile of the GATA-3-specific DNAzyme hgd40 after inhalation exposure

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.

Structural evolution and functional diversification analyses of argonaute protein

Argonaute (AGO) proteins are highly specialized small-RNA-binding modules and small RNAs are anchored to their specific binding pockets guiding AGO proteins to target mRNA molecules for silencing or destruction. The 135 full-length AGO protein sequences derived from 36 species covering prokaryote, archaea, and eukaryote are chosen for structural and functional analyses. The results show that bacteria and archaeal AGO proteins are clustered in the same clade and there exist multiple AGO proteins in most eukaryotic species, demonstrating that the increase of AGO gene copy number and horizontal gene transfer (HGT) have been the main evolutionary driving forces for adaptability and biodiversity. And the emergence of PAZ domain in AGO proteins is the unique evolutionary event. The analysis of middle domain (MID)-nucleotide contaction shows that either the position of sulfate I bond in Nc_QDE2 or the site of phosphate I bond in Hs_AGO2 represents the 5'-nucleotide binding site of miRNA. Also, H334, T335, and Y336 of Hs_AGO1 can form hydrogen bonds with 3'-overhanging ends of miRNAs and the same situation exists in Hs_AGO2, Hs_AGO3, Hs_AGO4, Dm_AGO1, and Ce_Alg1. Some PIWI domains containing conserved DDH motif have no slicer activity, and post-translational modifications may be associated with the endonucleolytic activities of AGOs. With the numbers of AGO genes increasing and fewer crystal structures available, the evolutionary and functional analyses of AGO proteins can help clarify the molecular mechanism of function diversification in response to environmental changes, and solve major issues including host defense mechanism against virus infection and molecular basis of disease.

Toward Systems Biology of Pulmonary Hypertension

In this issue of Circulation , Parikh and colleagues1 present an elegant study that lies at the intersection of 2 recent major developments in cardiovascular research—the recognition of the role of microRNAs in normal and abnormal cardiovascular biology, and the introduction of computational systems biology approaches to elucidate the mechanisms of cardiovascular disease. This work provides convincing evidence for the central role of microRNA-21 (miR-21) in the pathogenesis of pulmonary hypertension (PH) generally and pulmonary arterial hypertension (PAH) more specifically, and suggests a potential 2-hit rationale for incomplete penetrance of mutations in BMPR2 in heritable PAH. Article see p 1520 MicroRNAs are small noncoding RNA molecules 17 to 23 nucleotides long that bind to target mRNA molecules and repress their translation by cleavage, sequestration, degradation, or ribosomal inhibition. There are >1000 microRNAs in the genome, regulating ≈30% of gene expression.2 Although they were initially discovered in Caenorhabditis elegans in 1993, their role in mammalian cardiovascular biology has been recognized only in the last decade. Global deletion of functional microRNAs in smooth muscle cells in mice leads to impairment of vascular smooth muscle cell development, differentiation, and contraction, abrogated aortic development, extensive hemorrhage, and embryonic lethality.3 Similar deletion of microRNAs in endothelial cells is compatible with life, but leads to impaired angiogenesis.4 Thus, microRNAs are essential for vascular development and function. Further work, some of which is discussed below, has shown that dysregulation of microRNAs is associated with vascular disease. To establish the role of miR-21 in PH, Parikh and colleagues used an innovative approach integrating network analysis of existing genome-wide mRNA expression data, algorithmic prediction of microRNA targets, previously established concepts of biological pathways involved in PH, and in vitro and in vivo experimentation. First, they established a consolidated interactome of molecular interactions known to occur …

P3-03-06: Regulation of microRNA Expression by Autocrine Human Growth Hormone in Breast Cancer Cells.

Abstract Introduction: MicroRNAs (miRNAs) are a class of small non-protein coding RNAs approximately 20–24 nucleotides in length which regulate the expression of genes by pairing with the 3’ untranslated regions of target mRNA molecules and inhibiting translation or promoting mRNA degradation. Mature miRNAs are processed from longer transcripts by the miRNA biogenesis machinery, which includes key enzymes Dicer, Drosha and AGO-2. Altered miRNA expression has been implicated in many pathologies, including breast cancer. In general it is the deregulated expression of individual miRNAs that has been implicated in breast cancer pathogenesis. However, global downregulation of all or the majority of miRNAs has also been observed and can lead to a more invasive phenotype and increased proliferation in various cancers. Global miRNA downregulation has been associated with changes in expression levels of proteins involved in miRNA biogenesis. The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development. Studies investigating hGH expression in human breast cancer have demonstrated that hGH expression is positively correlated with lymph node metastasis, tumour stage, HER-2 status and proliferative index. In mammary carcinoma cells autocrine hGH promotes cell proliferation, survival, migration/invasion and epithelial-to-mesenchymal transition as well as tumour formation in a xenograft model. In the current study we demonstrate that autocrine hGH regulates miRNA biogenesis in breast cancer cells. Methods: Forced expression of hGH was established in the mammary carcinoma cell lines MCF-7 and T47D by stable transfection. miRNA expression was determined using miRNA microarray, quantitative real-time PCR (qPCR) arrays and qPCR assays. The expression levels of genes and proteins involved in miRNA biogenesis were assessed using real-time qPCR assays and Western blotting. Results: More than 90% of miRNAs assayed were downregulated in hGH-transfected MCF-7 and T47D cells, compared to control transfected cells, as demonstrated by miRNA microarray and qPCR array. Changes in miRNA expression determined by microarray and qPCR array were verified using miRNA-specific qPCR assays. In MCF-7 cells, autocrine hGH did not significantly affect the mRNA expression levels of miRNA machinery components Drosha, DGCR8, PACT, TARBP, EXP-5, Dicer and AGO2 when compared to control transfected cells. Whereas in T47D cells autocrine hGH increased mRNA expression of TARBP, PACT and EXP-5 by 2.0, 2.3 and 1.8 fold respectively, compared to control transfected cells. Western blot analysis demonstrated that, autocrine hGH decreased protein levels of Dicer in MCF-7 cells, whereas Drosha and AGO2 were unchanged compared to control transfected cells. In T47D cells autocrine hGH decreased protein levels of Dicer, Drosha and AGO2 when compared to control transfected cells. Conclusion: Our findings demonstrate that autocrine hGH stimulates global downregulation of miRNA expression in breast cancer cells. This may be one mechanism whereby autocrine hGH promotes tumour progression. Our results also indicate that autocrine hGH-mediated global downregulation of miRNA expression may occur through regulation of proteins involved in the miRNA biogenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-06.

Detours on the Road to Personalized Medicine

HE SEQUENCING OF THE HUMAN GENOME, THE ABILitytoperformgenome-wideassociationstudies,and thedevelopmentofhigh-throughputnucleotidesequencing support the goals of personalized medicine. “Next generation” sequencing platforms now providemuchhigherresolutionofDNAthanwaspossiblewith microarray-based–single nucleotide polymorphism (SNP) detection.Otherbreakthroughtechnologiessuchastheability to sequence DNA from single cells, targeted mRNA molecules,andmethylatedregionsofDNA;DNAfragmentlength analysis; isolation of circulating tumor cells; and the ability to use paraffin-embedded tissue for mRNA sequencing will make genotyping faster, easier, less expensive, better, and ultimately more readily available. The explosion in biomarker discovery now taking place in academic and biotech communities has exposed a new obstacle on the road to personalized medicine—neither the research nor the health care ecosystems have evolved capabilities for high-volume validation of potentially useful biomarkers in large populations of patients. The limitationsoftheexistingecosystemaredescribedintermsofbiomarker complexity, clinical research infrastructure, and reimbursement issues.

Viruses and microRNAs: a toolbox for systematic analysis

RNA silencing is emerging as a novel layer of regulation of virus-host interaction. Since individual small RNAs can probably repress dozens if not hundreds of target mRNA molecules, and transcripts, on the other hand, may be recognized by multiple regulatory small RNAs, a dense and complex interaction network of microRNAs (miRNAs) and their targets arises. A comprehensive analysis of miRNA functions thus not only requires systematic approaches employing high-throughput technologies but also calls for the development of improved experimental technologies and a profound bioinformatic analysis. Integration of complementary approaches will enhance our understanding of the mutual regulation of virus and host. Focusing on herpesviruses, we here describe currently available technologies and summarize present results obtained by high-throughput approaches. These techniques can be broadly applied to other virus families and pathways employing other classes of small regulatory RNAs and therefore are powerful universal tools for research on virus-host interaction.

RNA Interference and its therapeutic applications

RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA) trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC) and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS); other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans.

Fluorescence Correlation Spectroscopy Elucidates the Pathway of RNA Interference

Short double-stranded RNA molecules have recently emerged as important regulators of gene expression. These small RNAs associate with a member of the Argonaute protein family in an assembly known as RNA-induced silencing complex (RISC).Here we elucidate the pathway of RNA Interference (RNAi) in vivo by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS).We show that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex in the nucleus. Nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The import of Ago2 into the nucleus is mediated by the import receptor Impotin8.We further demonstrate that FCCS can be used to study the interaction of different members of the Argonaute protein family with short double-stranded RNAs and their target mRNA molecules.

Target mRNA abundance dilutes microRNA and siRNA activity

Post-transcriptional regulation by microRNAs and siRNAs depends not only on characteristics of individual binding sites in target mRNA molecules, but also on system-level properties such as overall molecular concentrations. We hypothesize that an intracellular pool of microRNAs/siRNAs faced with a larger number of available predicted target transcripts will downregulate each individual target gene to a lesser extent. To test this hypothesis, we analyzed mRNA expression change from 178 microRNA and siRNA transfection experiments in two cell lines. We find that downregulation of particular genes mediated by microRNAs and siRNAs indeed varies with the total concentration of available target transcripts. We conclude that to interpret and design experiments involving gene regulation by small RNAs, global properties, such as target mRNA abundance, need to be considered in addition to local determinants. We propose that analysis of microRNA/siRNA targeting would benefit from a more quantitative definition, rather than simple categorization of genes as ‘target' or ‘not a target.' Our results are important for understanding microRNA regulation and may also have implications for siRNA design and small RNA therapeutics.

Significant inverse correlation of microRNA-150/MYB and microRNA-222/p27 in myelodysplastic syndrome

We investigated whether, in myelodysplastic syndromes (MDS), aberrant expression of miR-150/miR-221/miR-222 and their designated target mRNA molecules MYB, p27 and c-KIT may be involved in insufficient haematopoiesis. In a series of MDS ( n = 52), an aberrant increase of miR-150 was found only in MDS with associated del(5q) ( n = 9; p < 0.01). The mRNA expression of transcription factor MYB, the designated target of miR-150, was shown to correlate inversely with the miR-150 level. Acute leukaemia evolving from MDS ( n = 11) showed significantly decreased levels of miR-221 but not miR-222. We conclude that inhibition of proliferation via over-expressed miR-150 might contribute to myelodysplastic haematopoiesis in MDS-del(5q).

Genomic Profiling of MicroRNAs in Bladder Cancer: miR-129 Is Associated with Poor Outcome and Promotes Cell Death In vitro

microRNAs (miRNA) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Here, we profiled the expression of 290 unique human miRNAs in 11 normal and 106 bladder tumor samples using spotted locked nucleic acid-based oligonucleotide microarrays. We identified several differentially expressed miRNAs between normal urothelium and cancer and between the different disease stages. miR-145 was found to be the most down-regulated in cancer compared with normal, and miR-21 was the most up-regulated in cancer. Furthermore, we identified miRNAs that significantly correlated to the presence of concomitant carcinoma in situ. We identified several miRNAs with prognostic potential for predicting disease progression (e.g., miR-129, miR-133b, and miR-518c*). We localized the expression of miR-145, miR-21, and miR-129 to urothelium by in situ hybridization. We then focused on miR-129 that exerted significant growth inhibition and induced cell death upon transfection with a miR-129 precursor in bladder carcinoma cell lines T24 and SW780 cells. Microarray analysis of T24 cells after transfection showed significant miR-129 target down-regulation (P = 0.0002) and pathway analysis indicated that targets were involved in cell death processes. By analyzing gene expression data from clinical tumor samples, we identified significant expression changes of target mRNA molecules related to the miRNA expression. Using luciferase assays, we documented a direct link between miR-129 and the two putative targets GALNT1 and SOX4. The findings reported here indicate that several miRNAs are differentially regulated in bladder cancer and may form a basis for clinical development of new biomarkers for bladder cancer.

All-in-One Tube Method for Quantitative Gene Expression Analysis in Oligo-dT30 Immobilized PCR Tube Coated with MPC Polymer

In this report, we have developed a novel quantitative RT-PCR protocol in which the procedure including mRNA purification can be performed in an all-in-one tube. To simplify gene expression analysis, oligo-dT(30) immobilized PCR tubes were used serially to capture mRNA, synthesize solid-phase cDNA, and amplify specific genes. The immobilized oligo-dT(30) can efficiently capture mRNA directly from crude human cell lysates. The captured mRNA is then amplified by one-step reverse transcription PCR (RT-PCR) with initial cDNA synthesis followed by PCR. In RT-PCR, this new reusable PCR tube device can be employed for multiple PCR amplifications with different primer sets from a solid-phase oligo-dT(30) primed cDNA library. This paper introduces a novel and highly reliable all-in-one tube method for rapid cell lysis, followed by quantitative preparation and expression analysis of target mRNA molecules with small amounts of sample. This procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction. We demonstrate the utility of this novel method by quantification of two housekeeping genes, beta-actin and GAPDH, in HeLa cells. We believe this new PCR device can be useful as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.

144. ROLE OF RNA-BINDING PROTEIN, MUSASHI-1 (Msi-1), IN MURINE FOLLICULOGENESIS AND OOCYTE DEVELOPMENT

Follicular development and oocyte maturation in mammals requires the temporal and spatial control of protein production. Consequently, it is hypothesised that the preovulatory follicle represses mRNA translation until specific proteins are required during oocyte maturation. Increasingly RNA-binding proteins are being recognised as important contributors to germ cell development, particularly during oocyte transcriptional quiescence. We have identified the presence of RNA-binding protein musashi-1 (Msi-1) mRNA within the mouse ovary and mature mouse oocyte, where the protein is believed to act as a translational repressor by binding to specific sequences within the 3' UTR of target mRNA molecules. Recent studies in various mammalian systems have identified p21 WAF1, cdkn2a, notch and m-numb as potential targets of Msi-1. We have also identified morf4l1 as a potential target through preliminary pulldown and microarray analysis using a GST tagged Msi-1 recombinant protein. To further study these potential targets, a transgenic Msi-1 mouse was produced to overexpress the RNA-binding protein in the developing oocyte. Real time PCR, performed on intact ovaries of WT and Tg mice, has so far demonstrated a 1.5-fold increase in Msi-1 expression in tgMsi-1/+ ovaries, above WT ovary expression. Real time PCR analysis of Msi-1 target mRNA expression has also shown an overall increase in expression in the tgMsi-1/+ ovaries of p21 WAF1 (~2.5-fold), cdkn2a (~2-fold), and notch (~3-fold). However m-numb and morf4l1 do not appear to be targets of Msi-1 in the oocyte, with no significant difference in expression between the WT and tgMsi-1/+ ovaries analysed. Functional quantification of oocyte development reveals a significantly less oocytes produced from superovulated juvenile mice compared with wild type litter mates. Therefore, preliminary analysis suggests that Msi-1 may play a role in binding the transcripts of genes necessary for cell cycle regulation and chromatin remodelling, characteristic of meiotic progression and oocyte development.