Validation of microRNA Target Genes Using Luciferase Reporter assay and Western Blot Analysis.

MicroRNA s regulate gene expression by binding to the 3'untranslated region (UTR) of the mRNA of their target genes. Identification of microRNA target genes enables the determination of their functional role in the cells. A single microRNA can target multiple genes, all of which have a microRNA binding site in their 3' UTR. Putative target genes can be identified using target prediction software and gene expression analysis of microRNA expressing cells. The validation of the putative target genes is carried out using the luciferase reporter assay and western blot analysis. This chapter describes the protocol for using these techniques for validation of putative microRNA target genes.

Quantification of microRNAs in Cells and Tissues Using Stem-Loop RT PCR and qPCR.

MicroRNA s are small RNA molecules that regulate gene expression by binding to the 3' untranslated region of the mRNA of their target genes. MicroRNA expression is altered in medulloblastoma as compared to the normal brain and this alteration is often associated with the pathogenesis of this tumor. The quantification of microRNA expression is carried out using quantitative/real-time polymerase chain reaction (PCR). In this chapter, we describe the protocol for the quantification of microRNA s in medulloblastoma tissues and cultured cells. This is carried out in three steps: (1) Extraction of total RNA, (2) Stem-loop reverse-transcriptase PCR, and (3) quantitative PCR.

Centriole Remodeling during Spermiogenesis in Drosophila.

The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM) [1]. In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote [2-4]. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis [5, 6]. Here, we show that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction [7]. We found that the ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and that the PCL does not resemble a typical centriole. We also describe a new phenomenon of Poc1 enrichment of the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, we found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila.

Asterless Reduction during Spermiogenesis Is Regulated by Plk4 and Is Essential for Zygote Development in Drosophila.

Centrosome reduction is the decrease in centrosomal components during spermatid differentiation (spermiogenesis). It is one of several dramatic subcellular reorganizations that lead to spermatozoa formation common to a wide range of animals. However, the mechanism underlying centrosome reduction is unknown and its functions are unclear. Here, we show that in Drosophila melanogaster spermiogenesis, the quantity of centrosomal proteins is dramatically reduced; for example, Asterless (Asl) is reduced ∼500-fold and is barely detected in spermatozoa. Asl reduction is regulated through a subset of its domains by the master regulator of centriole duplication Plk4 and by the ubiquitin ligase that targets Plk4 for degradation: Slimb. When Asl reduction is attenuated by Asl overexpression, plk4 mutations, Plk4 RNAi, or Slimb overexpression, Asl levels are higher in spermatozoa, resulting in embryos with reduced viability. Significantly, overexpressing Plk4 and Asl simultaneously, or combining plk4 and slimb mutations, balances their opposing effects on Asl reduction, restoring seemingly normal fertility. This suggests that increased Asl levels cause the observed reduced fertility and not other pleotropic effects. Attenuation of Asl reduction also causes delayed development and a failure to form astral microtubules in the zygote. Together, we provide the first insight into a molecular mechanism that regulates centrosome reduction and the first direct evidence that centrosome reduction is essential for post-fertilization development.

MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1.

Medulloblastoma, a common pediatric malignant brain tumor consists of four molecular subgroups viz. WNT, SHH, Group 3 and Group 4. MiR-148a is over-expressed in the WNT subgroup tumors, which have the lowest incidence of metastasis and excellent survival among all medulloblastomas. MiR-148a was expressed either in a transient manner using a synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector in non-WNT medulloblastoma cell lines. Expression of miR-148a to levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential, invasion potential and tumorigenicity of medulloblastoma cells. MiR-148a expression in medulloblastoma cells brought about reduction in the expression of NRP1, a novel miR-148a target. Restoration of NRP1 expression in medulloblastoma cells was found to rescue the reduction in the invasion potential and tumorigenicity brought about by miR-148a expression. NRP1 is known to play role in multiple signaling pathways that promote tumor growth, invasion and metastasis. NRP1 expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. The tumor suppressive effect of miR-148a expression accompanied by the down-regulation of NRP1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas.

Atypical centrioles during sexual reproduction.

Centrioles are conserved, self-replicating, microtubule-based, 9-fold symmetric subcellular organelles that are essential for proper cell division and function. Most cells have two centrioles and maintaining this number of centrioles is important for animal development and physiology. However, how animals gain their first two centrioles during reproduction is only partially understood. It is well established that in most animals, the centrioles are contributed to the zygote by the sperm. However, in humans and many animals, the sperm centrioles are modified in their structure and protein composition, or they appear to be missing altogether. In these animals, the origin of the first centrioles is not clear. Here, we review various hypotheses on how centrioles are gained during reproduction and describe specialized functions of the zygotic centrioles. In particular, we discuss a new and atypical centriole found in sperm and zygote, called the proximal centriole-like structure (PCL). We also discuss another type of atypical centriole, the "zombie" centriole, which is degenerated but functional. Together, the presence of centrioles, PCL, and zombie centrioles suggests a universal mechanism of centriole inheritance among animals and new causes of infertility. Since the atypical centrioles of sperm and zygote share similar functions with typical centrioles in somatic cells, they can provide unmatched insight into centriole biology.

The origin of the second centriole in the zygote of Drosophila melanogaster.

Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like (PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in asterless mutant embryos that cannot form centrioles. The zygote's centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization.