BackgroundGenetic mutations in several ubiquitously expressed RNA splicing genes such as PRPF3, PRP31 and PRPC8, have been found to cause retina-specific diseases in humans. To understand this intriguing phenomenon, most studies have been focused on testing two major hypotheses. One hypothesis assumes that these mutations interrupt retina-specific interactions that are important for RNA splicing, implying that there are specific components in the retina interacting with these splicing factors. The second hypothesis suggests that these mutations have only a mild effect on the protein function and thus affect only the metabolically highly active cells such as retinal photoreceptors.Methodology/Principal FindingsWe examined the second hypothesis using the PRPF3 gene as an example. We analyzed the spatial and temporal expression of the PRPF3 gene in mice and found that it is highly expressed in retinal cells relative to other tissues and its expression is developmentally regulated. In addition, we also found that PRP31 and PRPC8 as well as snRNAs are highly expressed in retinal cells.Conclusions/SignificanceOur data suggest that the retina requires a relatively high level of RNA splicing activity for optimal tissue-specific physiological function. Because the RP18 mutation has neither a debilitating nor acute effect on protein function, we suggest that retinal degeneration is the accumulative effect of decades of suboptimal RNA splicing due to the mildly impaired protein.

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3’ UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3’ UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.

Background: Vertebrate trunk induction requires inhibition of bone morphogenetic protein (BMP) signaling, whereas vertebrate head induction requires concerted inhibition of both Wnt and BMP signaling. RNA binding proteins play diverse roles in embryonic development and their roles in vertebrate head development remain to be elucidated. Results: We first characterized the human RBM47 as an RNA binding protein that specifically binds RNA but not single-stranded DNA. Next, we knocked down rbm47 gene function in zebrafish using morpholinos targeting the start codon and exon-1/intron-1 splice junction. Down-regulation of rbm47 resulted in headless and small head phenotypes, which can be rescued by a wnt8a blocking morpholino. To further reveal the mechanism of rbm47's role in head development, microarrays were performed to screen genes differentially expressed in normal and knockdown embryos. epcam and a2ml were identified as the most significantly up-and down-regulated genes, respectively. The microarrays also confirmed up-regulation of several genes involved in head development, including gsk3a, otx2, and chordin, which are important regulators of Wnt signaling. Conclusions: Altogether, our findings reveal that Rbm47 is a novel RNA-binding protein critical for head formation and embryonic patterning during zebrafish embryogenesis which may act through a Wnt8a signaling pathway. Developmental Dynamics 242:1395-1404, 2013. V C 2013 Wiley Periodicals, Inc.Key words: RNA binding protein; rbm47; head development; zebrafish; gene regulation; gene knockdown; morpholino; microarray Key findings:Human RBM47 is localized to the nucleus and is capable of interacting with RNA. Morpholino-based rbm47 knockdown in zebrafish results in loss of or reduced head development. Rbm47 functions through a pathway involving Wnt8a signaling.

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.