MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that was recently implicated in miRNA processing. Here we show that human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript.

The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAILand AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.The SNAG family of zinc finger transcription factors in vertebrates include GFI-1A, GFI-1B, the insulinoma-associated protein IA-1, the homeobox protein GSH-1, and the SNAIL/SLUG family. These proteins play important roles in the regulation of development, stem cell self-renewal, and tumor progression (5, 22, 49). They share a common set of functional domains: a C-terminal DNA binding domain composed of five to seven Cys2-His2 zinc fingers and a highly conserved N-terminal repression domain designated SNAG. The SNAG motif was first identified from the GFI-1 protein and comprises the first 21 amino acid residues in the N terminus. The SNAG domain is a potent and transferable repression motif (22, 49). However, unlike other repression domains which are associated with zinc finger proteins, such as the KRAB domain and the BTB-POZ domain, whose mechanisms of repression are well established, little is known about the mechanisms of the SNAG domain-mediated repression (9, 15).The SNAIL protein has emerged as a potent regulator of the processes of embryonic development and tumor progression through the regulation of the epithelial-mesenchymal transition (EMT) (5, 36). In mammalian cells, SNAIL induces EMT at least partially through repression of the E-cadherin gene, thereby altering cell adhesion (6). The SNAIL protein has been found in multiprotein complexes containing histone deacetylases (HDACs), mSIN3A, and LOXL2/3 (39, 40). However, the biological significance of these interactions and how SNAIL mediates functional protein complex assembly at specific promoters in the context of chromatin remain undefined.We previously identified novel corepressors that directly bind to the SNAG domains of GFI-1 and SNAIL by using yeast t...

Geckos use vocalizations for intraspecific communication, but little is known about the organization of their central auditory system. We therefore used antibodies against the calcium-binding proteins calretinin (CR), parvalbumin (PV), and calbindin-D28k (CB) to characterize the gecko auditory system. We also examined expression of both glutamic acid decarboxlase (GAD) and synaptic vesicle protein (SV2). Western blots showed that these antibodies are specific to gecko brain. All three calcium-binding proteins were expressed in the auditory nerve, and CR immunoreactivity labeled the first-order nuclei and delineated the terminal fields associated with the ascending projections from the first-order auditory nuclei. PV expression characterized the superior olivary nuclei, whereas GAD immunoreactivity characterized many neurons in the nucleus of the lateral lemniscus and some neurons in the torus semicircularis. In the auditory midbrain, the distribution of CR, PV, and CB characterized divisions within the central nucleus of the torus semicircularis. All three calcium-binding proteins were expressed in nucleus medialis of the thalamus. These expression patterns are similar to those described for other vertebrates.

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WD40 repeat 1 protein (WDR1) was first reported in the acoustically injured chicken inner ear, and bioinformatics revealed that WDR1 has numerous WD40 repeats, important for protein-protein interactions. It has significant homology to actin interacting protein 1 (Aip1) in several lower species such as yeast, roundworm, fruitfly and frog. Several studies have shown that Aip1 binds cofilin/actin depolymerizing factor, and that these interactions are pivotal for actin disassembly via actin filament severing and actin monomer capping. However, the role of WDR1 in auditory function has yet to be determined. WDR1 is typically restricted to hair cells of the normal avian basilar papilla, but is redistributed towards supporting cells after acoustic overstimulation, suggesting that WDR1 may be involved in inner ear response to noise stress. One aim of the present study was to resolve the question as to whether stress factors, other than intense sound, could induce changes in WDR1 presence in the affected avian inner ear. Several techniques were used to assess WDR1 presence in the inner ears of songbird strains, including Belgian Waterslager (BW) canary, an avian strain with degenerative hearing loss thought to have a genetic basis. Reverse transcription, followed by polymerase chain reactions with WDR1-specific primers, confirmed WDR1 presence in the basilar papillae of adult BW, non-BW canaries, and zebra finches. Confocal microscopy examinations, following immunocytochemistry with anti-WDR1 antibody, localized WDR1 to the hair cell cytoplasm along the avian sensory epithelium. In addition, little, if any, staining by anti-WDR1 antibody was observed among supporting cells in the chicken or songbird ear. The present observations confirm and extend the early findings of WDR1 localization in hair cells, but not in supporting cells, in the normal avian basilar papilla. However, unlike supporting cells in the acoustically damaged chicken basilar papilla, the inner ear of the BW canary showed little, if any, WDR1 up-regulation in supporting cells. This may be due to the fact that the BW canary already has established hearing loss and/or to the possibility that the mechanism(s) involved in BW hearing loss may not be related to WDR1.