MicroRNAs play important roles in regulating development at both transcriptional and posttranscriptional levels. Here, we report 29 microRNAs from mouse testis that are differentially expressed as the prepubertal testis differentiates to the adult testis. Using computational analyses to identify potential microRNA target mRNAs, we identify several possible male germ cell target mRNAs. One highly conserved sequence in the 3'-untranslated region (UTR) of transition protein 2 (Tnp2) mRNA, a testis-specific and posttranscriptionally regulated mRNA in postmeiotic germ cells, is complementary to Mirn122a. Mirn122a is enriched in late-stage male germ cells and is predominantly on polysomes. Mirn122a, but not another noncomplementary microRNA, inhibits the activity of a luciferase reporter construct containing the 3'-UTR of Tnp2. Site-directed mutations of Mirn122a indicate that base pairing of the 5'-region of Mirn122a to its complementary site in the 3'-UTR of Tnp2 mRNA is essential for the downregulation of luciferase activity. Real-time reverse transcription-polymerase chain reaction and ribonuclease protection assays reveal that the Mirn122a-directed decrease of the Tnp2 reporter gene activity results from mRNA cleavage. We propose that specific microRNAs, such as Mirn122a, could be involved in the posttranscriptional regulation of mRNAs such as Tnp2 in the mammalian testis.

Two chemokine (chemoattractant cytokines) fJ peptides, macrophage inflammatory proteins la and lip

Poly(A) polymerase has a critical role in the synthesis of messenger RNA in eukaryotic cells. The isolation and characterization of complementary DNAs encoding bovine poly(A) polymerase is described here. The predicted sequences of the mRNA and protein reveal features that provide insights into how the enzyme functions and how it might be regulated. Poly(A) polymerase expressed from a cloned cDNA is fully functional in in vitro assays, and mutational analyses have identified a putative regulatory domain that enhances, but is not essential for, activity.

The synthesis of tumor necrosis factor-␣ has been suggested to be regulated at both the transcriptional and translational levels in response to stimulation by bacterial lipopolysaccharide, although the relative contribution of these two mechanisms has not been quantitatively evaluated. Here, using the murine monocytic cell line RAW 264.7 as a model system, we show that steadystate TNF-␣ mRNA levels increase ϳ77-fold following treatment with lipopolysaccharide for 2 h and to a maximum of 164-fold after 8 h as measured by an RNase protection assay. The TNF-␣ gene transcription rate increases ϳ5-fold following exposure to lipopolysaccharide for 2 h as measured by a nuclear run-on assay. TNF-␣ mRNA stability did not change in the presence of lipopolysaccharide. A ribosomal sedimentation assay and an RNA transfection assay revealed that the translation rate of endogenous as well as transiently transfected TNF-␣ mRNAs increases only ϳ2-3-fold after stimulation with lipopolysaccharide for 2 h. Taken together, these results suggest that the large increase in the level of secreted TNF-␣ protein in RAW 264.7 cells is due primarily to activation of TNF-␣ gene transcription.

Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors and studied the precursor–product relationships that linked the subsets identified. This analysis identified two successive stages of ILC development within TCF-1 + early innate lymphoid progenitors (EILPs), which we named ‘specified EILPs’ and ‘committed EILPs’. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILP. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate.