Identification of MIWI-associated Poly(A) RNAs by immunoprecipitation with an anti-MIWI monoclonal antibody.

MIWI is one of the PIWI subfamily of proteins mainly expressed in mouse germ cells, and associates with pachytene piRNAs. MIWI has been thought to play an essential role in spermatogenesis and spermiogenesis via biogenesis and/or stability of pachytene piRNAs, retrotransposon silencing, and post-transcriptional regulation of target mRNAs. However, MIWI's detailed role and function are not well understood. In this study, we produced an anti-MIWI mouse monoclonal antibody and identified MIWI-associated poly(A) RNAs by immunoprecipitation from adult mouse testes lysates. Approximately 70% of the MIWI-associated poly(A) RNAs were known mRNAs and 30% of them were unknown non-coding RNAs. These poly(A) RNAs contained piRNA-encoding RNAs transcribed from piRNA cluster regions and piRNA-encoding mRNA, such as Aym1 mRNA. Mature piRNAs specifically encoded in these piRNA-encoding RNAs were generated in pachytene spermatocytes and not detected in Miwi-deficient (Miwi-/-) testes. Moreover, MIWI associated with a large number of known mRNAs whose expression levels were increased in pachytene spermatocytes, and the expression of these mRNAs was decreased in Miwi-/- testes at 20 days postpartum when pachytene spermatocytes were most abundant. These results strongly suggest that MIWI is involved in pachytene piRNA biogenesis and the positive regulation of target mRNA metabolism in pachytene spermatocytes via association with pachytene piRNA precursors and target mRNAs.

A sensitive sandwich ELISA to measure (1→3)-ß-d-glucan levels in blood.

A highly sensitive (1→3)-ß-d-glucan (ß-glucan)-specific sandwich ELISA was developed using a fragment of recombinant horseshoe crab factor G protein. The factor G fragment, which was expressed in Escherichia coli, contains a QQWS motif, two ß-glucan-binding domains, and an additional N-terminal cysteine residue. The sensitivity of our ELISA was comparable to a conventional (1→3)-ß-d-glucan detection method using a horseshoe crab-clotting reaction such as an amebocyte lysate-based assay. In addition, the ß-glucan levels measured by our sandwich ELISA in plasma samples showed a good correlation with those measured by the amebocyte lysate-based assay. In the case of our sandwich ELISA, it is not necessary to pre-inactivate interfering substances in plasma samples that is essential for the conventional amebocyte lysate-based assay. Moreover, the assay time of the ELISA method is much shorter than that of the amebocyte lysate-based assay. Because of these advantages, the ELISA system will be more suitable for high-throughput analysis in clinical laboratories using general clinical auto-analyzers. ß-glucan is a typical biomarker for fungal infections and the measurements of ß-glucan levels by our ELISA could be useful for the diagnosis of fungal infections.

A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

A useful approach to total analysis of RISC-associated RNA.

BACKGROUND: Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC. FINDINGS: With our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 107 cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs. CONCLUSION: Our method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.

Intracerebroventricular administration of urotensin II promotes anxiogenic-like behaviors in rodents.

We identified urotensin II (U-II) as the endogenous ligand for the orphan G-protein-coupled receptor GPR14 or SENR. Both U-II and GPR14 are expressed not only in peripheral tissues but also in the brain of rodents, suggesting that U-II plays a physiological role in the central nervous system. In the present study, we investigated the central effects of U-II in rodents. Intracerebroventricular administration of U-II induced anxiogenic-like behaviors in the elevated plus maze test and the hole-board test in mice in a dose-dependent manner, as did corticotropin releasing factor (CRF). The effective doses of U-II were 10-100-fold higher than these of CRF in these tests. Our results suggest that U-II is a candidate for the mediator of some aspect of stress or anxiety in the central nervous system.

Galanin-like peptide stimulates food intake in the rat.

We have isolated a novel hypothalamic peptide, Galanin-like peptide (GALP), as a ligand for galanin receptor subtype GalR2. To investigate the physiological role of GALP, we examined the effect of the intracerebroventricular administration of GALP and found that GALP induced food intakes. GALP had ten-fold the orexigenic activity of galanin. We also observed the anxiogenic-like behavior after the administration of 1 nmol GALP. These results suggest that GALP is a novel orexigenic and anxiogenic peptide.