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The differentiation of naive CD4(+) T cells into lineages with distinct effector functions has been considered to be an irreversible event. T helper type 1 (Th1) cells stably express IFN-gamma, whereas Th2 cells express IL-4. The discovery and investigation of two other CD4(+) T cell subsets, induced regulatory T (iTreg) cells and Th17 cells, has led to a rethinking of the notion that helper T cell subsets represent irreversibly differentiated endpoints. Accumulating evidence suggests that CD4(+) T cells, particularly iTreg and Th17 cells, are more plastic than previously appreciated. It appears that expression of Foxp3 by iTreg cells or IL-17 by Th17 cells may not be stable and that there is a great degree of flexibility in their differentiation options. Here, we will discuss recent findings that demonstrate the plasticity of CD4(+) T cell differentiation and the biological implications of this flexibility.

The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3′ end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development.Argonaute proteins are the key effectors of small RNA-mediated regulatory pathways that modulate gene expression, regulate chromosome structure and function, and provide an innate immune defence against viruses and transposons 1 . The structure of Ago proteins is well conserved, consisting of an amino-terminal domain, the Mid domain, and their signature PAZ and Piwi domains. Structure-function relationships in this family are becoming increasingly well understood 2 . The PAZ and Mid domains help to anchor the small RNA guide, with PAZ binding the 3′ end using a series of conserved aromatic residues and the Mid domain providing a binding pocket for the 5′ end. The Piwi domain contains an RNase H motif that was cryptic in the primary sequence but easily recognizable in the tertiary structure. Loading of a highly complementary target into an Ago brings the scissile ©2010 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to G.J.H. (hannon@cshl.edu). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions S.C. and G.J.H. planned experiments, interpreted data and wrote the paper. S.C. and C.O.D.S. performed studies, and M.M.W.C. provided critical unpublished reagents and discussion.Author Information Sequencing data has been deposited in GEO and assigned accession number GSE21370. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 December 1. Animal microRNAs function differently from their plant counterparts, with nearly all microRNA-target interactions providing insufficient complementarity to orient the ...

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell death. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

MicroRNAs (miRNAs) are implicated in the differentiation and function of many cell types. We provide genetic and in vivo evidence that the two RNaseIII enzymes, Drosha and Dicer, do indeed function in the same pathway. These have previously been shown to mediate the stepwise maturation of miRNAs (Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim. 2003. Nature. 425:415–419), and genetic ablation of either within the T cell compartment, or specifically within Foxp3+ regulatory T (T reg) cells, results in identical phenotypes. We found that miRNA biogenesis is indispensable for the function of T reg cells. Specific deletion of either Drosha or Dicer phenocopies mice lacking a functional Foxp3 gene or Foxp3+ cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease, but later in life. Thus, miRNA-dependent regulation is critical for preventing spontaneous inflammation and autoimmunity.

The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer-and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency.[Keywords: RNaseIII enzymes; microRNA targets; microRNA processing; endonucleolytic cleavage] Supplemental material is available at http://www.genesdev.org.

The lifespan of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here, we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5′-phosphate groups in mouse ES. We detect a prominent signature of Ago2-dependent cleavage events and validate several such targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mES cells, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.