Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

<p>Abstract</p> <p>Background</p> <p>Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.</p> <p>Results</p> <p>By RT PCR and western blot analysis we found that nucleolin is strongly down regulated in meiotic spermatocytes and haploid germ cells. We have identified a new nucleolin related protein (NRP) gene in the rat genome, which is over expressed in the testis and is up regulated several fold in meiotic spermatocytes and haploid germ cells. The NRP protein lacks the acidic stretches in its N terminal domain, and it is encoded in rat chromosome 15 having a different genomic organization as compared to nucleolin gene present on chromosome 9. We have also found NRP genes encoded in genomes of other mammalian species. We performed run-on transcription assay where we have observed that rDNA is transcribed at much lower level in meiotic spermatocytes and haploid spermatids as compared to diploid cells. By siRNA knock down experiments we could also demonstrate that NRP can support rDNA transcription in the absence of nucleolin.</p> <p>Conclusion</p> <p>We have identified a new nucleolin variant over expressed in germ cells in rat and analyzed its domain structure. We attribute that the transcriptional activity of rDNA genes in the late spermatogenesis is due to the presence of this variant NRP. The expression of this variant in the germ cells in the absence of nucleolin, could have additional functions in the mammalian spermatogenesis which needs to be investigated further.</p

Not Your Average Cadherin: Optimization of Expression, Purification, and Initial Structural Analysis of the Atypical Celsr1

Cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1) is an atypical cadherin that is central to the planar cell polarity (PCP) pathway. The PCP pathway organizes polarization across the tissue plane, and is involved in a myriad of biological functions, from patterning stereocilia in the inner ear to organizing neural tube closure during fetal development. When this pathway malfunctions, various health conditions can arise, such as congenital heart disease, neural tube defects, and cancer. Celsr1 binds to itself in trans to form bridges between adjacent cells at cell-cell junctions, and asymmetrically recruits fellow membrane proteins Frizzled6 and Vangl2 to propagate polarization. Although Celsr1 is a critical part of an important biological pathway, very little is known about its structure, binding mechanism, or even the ratios in which it binds itself, Frizzled6, and Vangl2. Determining a high-resolution structure of Celsr1 should elucidate the residues involved in its homophilic binding activity, providing insight into its mechanism of binding and potentially mechanisms of inhibition, which would further understanding of Celsr1 and the PCP pathway as a whole. This thesis focuses on optimizing expression and purification of recombinant Celsr1 for sample preparation for cryogenic electron microscopy (cryo-EM), and analysis of initial structural data obtained from imaging prepared samples. Determining the optimal construct, expression system, and purification protocol resulted in enough highly concentrated pure protein to create a low-resolution map of Celsr1, which with further data collection should yield a high resolution solved structure. This thesis represents exciting steps on the path to solving the structure of Celsr1

A novel noncoding RNA processed by Drosha is restricted to nucleus in mouse

Noncoding RNAs constitute a huge repertoire of gene regulatory molecules. Our previous, fine-resolution characterization of a mouse meiotic recombination hotspot from chromosome 8 resulted in identification of 2.4-kb unspliced and polyadenylated noncoding mrhl RNA. The gene is expressed in multiple tissues and is also present in rat but absent in humans. Here we report that the mrhl RNA gets processed to a small 80-nucleotide (nt) RNA species and is mediated by the Drosha complex. We also observe that the 80-nt Drosha product could be processed further to a 22-nt small RNA by Dicer in an in vitro reaction. However, this 22-nt product was not detected in vivo. The 80-nt as well as the 2.4-kb full-length RNA are nuclear-localized, showing distinct punctate nuclear signal. The colocalization of the noncoding RNA with Drosha and Nucleolin suggests the nucleolus as the site of processing of the 2.4-kb primary transcript. Additional foci of the processed 80-nt RNA were also observed outside the nucleolus, suggesting its role in some specific chromatin domain(s). Thus, this study reports a novel noncoding mrhl RNA that is processed and restricted within the cell nucleus