Analysis of male sterile mutations in the mouse using haploid stage expressed cDNA probes

A differential hybridization screening procedure has identified cDNAs which correspond to RNAs which are expressed in mouse testis and at lower levels in liver and spleen. The sensitivity of this procedure is such that approximately 0.5% of 1.4 X 10(4) cDNA clones are revealed as "testis specific". We have focused on ten cDNA clones which have been used to identify RNAs expressed in the haploid phase of spermatogenesis. Using Northern blots to analyse RNA isolated from the testes of mutant mice (Tfm/Y and Sxr/+) blocked at specific stages in spermatogenesis or RNA from sexually immature mice, 8 clones have been identified which correspond to RNAs expressed uniquely or at much higher levels in meiotic or post meiotic cells.     

Induction of human aquaporin-1 gene by retinoic acid in human erythroleukemia HEL cells

Retinoids have been implicated in the control of cell proliferation, differentiation, and developmental processes. We report here that aquaporin-1 (AQP1) is specifically induced by retinoic acid (RA) in human erythroleukemia HEL cells. Both all-trans-RA (ATRA) and 9-cis-RA (9CRA) strongly induced the AQP1 mRNA and protein in a dose-dependent manner. AQP1 protein was mainly expressed in plasma membrane in cells induced by RAs. To identify the RA response element (RARE) in the human AQP1 promoter, the 5(')-flanking region of AQP1 promoter was isolated and transient transfection experiment in HEL cells was performed. Deletion analysis of the AQP1 promoter revealed that one putative DR5-like RARE with five spaces was located in the region from -2218 to -2202; AGGGCAgggacAGGTGA. Electrophoretic mobility shift assay (EMSA) experiment demonstrated that two slowly migrated complexes (C1 and C2) capable of binding the RARE sequence were present in nuclear extracts prepared from cells and the complex C1 was strongly increased in nuclear extracts by RA stimulation. The complexes C1 and C2 were significantly abolished by an excess unlabeled probe. These results indicate that RAs strongly stimulate the human AQP1 gene expression through the RARE and define a novel role in the regulation of erythropoiesis.     

Mice deficient for a small cluster of Piwi-interacting RNAs implicate Piwi-interacting RNAs in transposon control

The mammalian testis expresses a class of small noncoding RNAs that interact with mammalian PIWI proteins. In mice, the PIWI-interacting RNAs (piRNAs) partner with mammalian PIWI proteins, PIWIL1 and PIWIL2, also known as MIWI and MILI, to maintain transposon silencing in the germline genome. Here, we demonstrate that inactivation of Nct1/2, two noncoding RNAs encoding piRNAs, leads to derepression of LINE-1 (L1) but does not affect mouse viability, spermatogenesis, testicular gene expression, or fertility. These findings indicate that piRNAs from a cluster on chromosome 2 are necessary to maintain transposon silencing.     

Cloning and expression profiling of small RNAs expressed in the mouse ovary

Small noncoding RNAs have been suggested to play important roles in the regulation of gene expression across all species from plants to humans. To identify small RNAs expressed by the ovary, we generated mouse ovarian small RNA complementary DNA (srcDNA) libraries and sequenced 800 srcDNA clones. We identified 236 small RNAs including 122 microRNAs (miRNAs), 79 piwi-interacting RNAs (piRNAs), and 35 small nucleolar RNAs (snoRNAs). Among these small RNAs, 15 miRNAs, 74 piRNAs, and 21 snoRNAs are novel. Approximately 70% of the ovarian piRNAs are encoded by multicopy genes located within the repetitive regions, resembling previously identified repeat-associated small interference RNAs (rasiRNAs), whereas the remaining approximately 30% of piRNA genes are located in nonrepetitive regions of the genome with characteristics similar to the majority of piRNAs originally cloned from the testis. Since these two types of piRNAs display different structural features, we categorized them into two classes: repeat-associated piRNAs (rapiRNAs, equivalent of the rasiRNAs) and non-repeat-associated piRNAs (napiRNAs). Expression profiling analyses revealed that ovarian miRNAs were either ubiquitously expressed in multiple tissues or preferentially expressed in a few tissues including the ovary. Ovaries appear to express more rapiRNAs than napiRNAs, and sequence analyses support that both may be generated through the "ping-pong" mechanism. Unique expression and structural features of these ovarian small noncoding RNAs suggest that they may play important roles in the control of folliculogenesis and female fertility.