Novrouz B. Akhmedov

Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles

Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21–24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches.

A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors.

Abstract. C57BL/6J-c2J (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J × BALB/c)F1× c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c.When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

Differential Expression of Rod Photoreceptor cGMP-Phosphodiesterase α and β Subunits mRNA AND PROTEIN LEVELS

The catalytic core of photoreceptor-specific cGMP-phosphodiesterase (PDE) consists of two subunits, PDEα and PDEβ, that are homologous and have similar domain organization but are encoded by different genes. We have examined the PDEα and PDEβ mRNA steady-state and protein levels as well as the biosynthesis rate of these proteins in developing and fully differentiated retinas. We have also determined the translational efficiency of PDE subunits and the role of their mRNA structures in regulating protein synthesis. In mature retinas, PDEα and PDEβ are represented by ∼1.5 × 108 and 7.5 × 108 copies/μg retinal mRNA, respectively. The levels of these transcripts in developing photoreceptors (P10) are approximately 75% of those at P30. Quantification of protein concentration indicated that PDEα and PDEβ are equally expressed in developing and fully differentiated photoreceptors. Furthermore, the PDEα/PDEβ ratios obtained throughout a 2-h pulsechase period revealed a similar turnover rate for both subunits. The observed discordance between the mRNA and protein levels of PDEα and PDEβ suggested post-transcriptional regulation of their expression. We found that PDEα mRNA is translated more efficiently than either of the two PDEβ transcripts expressed in retina. Therefore, the lower level of PDEα mRNA is compensated by its more efficient translation to achieve equimolar expression with PDEβ. We also analyzed the effect of PDEα and PDEβ mRNA 5′- and 3′-untranslated regions as well as that of their coding regions on protein synthesis. We determined that the PDE-coding regions play a critical role in the differential translation of these subunits.

ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

PURPOSE To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina. METHODS The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis. RESULTS One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors. CONCLUSIONS ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.

ZBED4, A Novel Retinal Protein Expressed in Cones and Müller Cells

To identify genes expressed in cone photoreceptors, we previously carried out subtractive hybridization and microarrays of retinal mRNAs from normal and cd (cone degeneration) dogs. One of the isolated genes encoded ZBED4, a novel protein that in human retina is localized to cone photoreceptors and glial Müller cells. ZBED4 is distributed between nuclear and cytoplasmic fractions of the retina and it readily forms homodimers, probably as a consequence of its hATC dimerization domain. In addition, the ZBED4 sequence has several domains that suggest it may function as part of a co-activator complex facilitating the activation of nuclear receptors and other factors (BED finger domains) or as a co-activator/co-repressor of nuclear hormone receptors (LXXLL motifs). We have identified several putative ZBED4-interacting proteins and one of them is precisely a co-repressor of the estrogen receptor alpha.