mRNA-LNP vaccine-induced CD8+ T-cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T-cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T-cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T-cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T-cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T-cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T-cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Azobenzene-based colorimetric and fluorometric chemosensor for nitroxyl releasing.

The precise release and characterization of nitroxyl (HNO) gas signaling molecule remain a challenge due to its short lifetime to date. To solve this issue, an azobenzene-based HNO donor (Azo-D1) was proposed as a colorimetric and fluorometric chemosensor for HNO releasing, to release both HNO and an azobenzene fluorescent reporter together. Specifically, the Azo-D1 has an HNO release half-life of ∼68 min under physiological conditions. The characteristic color change from the original orange to the yellow color indicated the decomposition of the donor molecule. In addition, the stoichiometry release of HNO was qualitatively and quantitatively verified through the classical phosphine compound trap. As compared with the donor molecule by itself, the decomposed product demonstrates a maximum fluorescence emission at 424 nm, where the increase of fluorescence intensity by 6.8 times can be applied to infer the real-time concentration of HNO. Moreover, cellular imaging can also be achieved using this Azo-D1 HNO donor through photoexcitation at 405 and 488 nm, where the real-time monitoring of HNO release was achieved without consuming the HNO source. Finally, the Azo-D1 HNO donor would open a new platform in the exploration of the biochemistry and the biology of HNO.

Continuous In Vivo Monitoring of Indole-3-Acetic Acid and Salicylic Acid in Tomato Leaf Veins Based on an Electrochemical Microsensor.

Indole-3-acetic acid (IAA) and salicylic acid (SA), as critical plant hormones, are involved in multiple physiological regulatory processes of plants. Simultaneous and continuous in vivo detection of IAA and SA will help clarify the mechanisms of their regulation and crosstalk. First, this study reports the development and application of an electrochemical microsensor for simultaneous and continuous in vivo detection of IAA and SA. This electrochemical microsensor system consisted of a tip (length, 2 mm) of platinum wire (diameter, 0.1 mm) modified with carbon cement and multi-walled carbon nanotubes, an untreated tip (length, 2 mm) of platinum wire (diameter, 0.1 mm), as well as a tip (length, 2 mm) of Ag/AgCl wire (diameter, 0.1 mm). It was capable of detecting IAA in the level ranging from 0.1 to 30 µM and SA ranging from 0.1 to 50 µM based on the differential pulse voltammetry or amperometric i-t., respectively. The dynamics of IAA and SA levels in tomato leaf veins under high salinity stress were continuously detected in vivo, and very little damage occurred. Compared to conventional detection methods, the constructed microsensor is not only suitable for continuously detecting IAA and SA in microscopic plant tissue in vivo, it also reduces the damage done to plants during the detection. More importantly, the continuous and dynamic changes in IAA and SA data obtained in stiu through this system not only can help clarify the interaction mechanisms of IAA and SA in plants, it also helps to evaluate the health status of plants, which will promote the development of basic research in botany and precision agriculture.

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

Nitric oxide releasing polyvinyl alcohol and sodium alginate hydrogels as antibacterial and conductive strain sensors.

Conductive hydrogels have promising applications in flexible electronic devices and artificial intelligence, which have attracted much attention in recent years. However, most conductive hydrogels have no antimicrobial activity, inevitably leading to microbial infections during utilization. In this work, a series of antibacterial and conductive polyvinyl alcohol and sodium alginate (PVA-SA) hydrogels were successfully developed with the incorporation of S-nitroso-N-acetyl-penicillamine (SNAP) and MXene through a freeze-thaw approach. Due to the reversibility of hydrogen bonding and electrostatic interactions, the resulting hydrogels had excellent mechanical properties. Specifically, the presence of MXene readily interrupted the crosslinked hydrogel network, but the best stretching can reach up to >300 %. Moreover, the impregnation of SNAP achieved the release of nitric oxide (NO) over several days under physiological conditions. Due to the release of NO, these composited hydrogels demonstrated high antibacterial activities (> 99 %) against both Gram-positive and negative S. aureus and E. coli bacteria. Notably, the excellent conductivity of MXene endowed the hydrogel with a sensitive, fast, and stable strain-sensing ability, to accurately monitor and distinguish subtle physiological activities of the human body including finger bending and pulse beating. These novel composited hydrogels are likely to have potential as strain-sensing materials in the field of biomedical flexible electronics.

Resistance To Poxvirus Lethality Does Not Require the Necroptosis Proteins RIPK3 or MLKL.

Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins that are critical for necroptosis, a mechanism of programmed cell death that is both activated when apoptosis is inhibited and thought to be antiviral. Here, we investigated the role of RIPK3 and MLKL in controlling the Orthopoxvirus ectromelia virus (ECTV), a natural pathogen of the mouse. We found that C57BL/6 (B6) mice deficient in RIPK3 (Ripk3-/-) or MLKL (Mlkl-/-) were as susceptible as wild-type (WT) B6 mice to ECTV lethality after low-dose intraperitoneal infection and were as resistant as WT B6 mice after ECTV infection through the natural footpad route. Additionally, after footpad infection, Mlkl-/- mice, but not Ripk3-/- mice, endured lower viral titers than WT mice in the draining lymph node (dLN) at three days postinfection and in the spleen or in the liver at seven days postinfection. Despite the improved viral control, Mlkl-/- mice did not differ from WT mice in the expression of interferons or interferon-stimulated genes or in the recruitment of natural killer (NK) cells and inflammatory monocytes (iMOs) to the dLN. Additionally, the CD8 T-cell responses in Mlkl-/- and WT mice were similar, even though in the dLNs of Mlkl-/- mice, professional antigen-presenting cells were more heavily infected. Finally, the histopathology in the livers of Mlkl-/- and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. IMPORTANCE The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route. Mice deficient in MLKL are more efficient than WT mice at controlling virus loads in various organs. This improved viral control is not due to enhanced interferon, natural killer cell, or CD8 T-cell responses. Overall, the data indicate that deficiencies in the molecules that are critical to necroptosis do not necessarily result in worse outcomes following viral infection and may improve virus control.

Interferon partly dictates a divergent transcriptional response in poxvirus-infected and bystander inflammatory monocytes.

Inflammatory monocytes (iMOs) and B cells are the main targets of the poxvirus ectromelia virus (ECTV) in the lymph nodes of mice and play distinct roles in surviving the infection. Infected and bystander iMOs control ECTV's systemic spread, preventing early death, while B cells make antibodies that eliminate ECTV. Our work demonstrates that within an infected animal that survives ECTV infection, intrinsic and bystander infection of iMOs and B cells differentially control the transcription of genes important for immune cell function and, perhaps, cell identity. Bystander cells upregulate metabolism, antigen presentation, and interferon-stimulated genes. Infected cells downregulate many cell-type-specific genes and upregulate transcripts typical of non-immune cells. Bystander (Bys) and infected (Inf) iMOs non-redundantly contribute to the cytokine milieu and the interferon response. Furthermore, we uncover how type I interferon (IFN-I) or IFN-γ signaling differentially regulates immune pathways in Inf and Bys iMOs and that, at steady state, IFN-I primes iMOs for rapid IFN-I production and antigen presentation.

Mechanisms of Antiviral Cytotoxic CD4 T Cell Differentiation.

Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.

Resistance to lethal ectromelia virus infection requires Type I interferon receptor in natural killer cells and monocytes but not in adaptive immune or parenchymal cells.

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.

Lipid-nanoparticle-encapsulated mRNA vaccines induce protective memory CD8 T cells against a lethal viral infection.

It is well established that memory CD8 T cells protect susceptible strains of mice from mousepox, a lethal viral disease caused by ectromelia virus (ECTV), the murine counterpart to human variola virus. While mRNA vaccines induce protective antibody (Ab) responses, it is unknown whether they also induce protective memory CD8 T cells. We now show that immunization with different doses of unmodified or N(1)-methylpseudouridine-modified mRNA (modified mRNA) in lipid nanoparticles (LNP) encoding the ECTV gene EVM158 induced similarly strong CD8 T cell responses to the epitope TSYKFESV, albeit unmodified mRNA-LNP had adverse effects at the inoculation site. A single immunization with 10 µg modified mRNA-LNP protected most susceptible mice from mousepox, and booster vaccination increased the memory CD8 T cell pool, providing full protection. Moreover, modified mRNA-LNP encoding TSYKFESV appended to green fluorescent protein (GFP) protected against wild-type ECTV infection while lymphocytic choriomeningitis virus glycoprotein (GP) modified mRNA-LNP protected against ECTV expressing GP epitopes. Thus, modified mRNA-LNP can be used to create protective CD8 T cell-based vaccines against viral infections.

Viral infection modulates Qa-1b in infected and bystander cells to properly direct NK cell killing.

Natural killer (NK) cell activation depends on the signaling balance of activating and inhibitory receptors. CD94 forms inhibitory receptors with NKG2A and activating receptors with NKG2E or NKG2C. We previously demonstrated that CD94-NKG2 on NK cells and its ligand Qa-1b are important for the resistance of C57BL/6 mice to lethal ectromelia virus (ECTV) infection. We now show that NKG2C or NKG2E deficiency does not increase susceptibility to lethal ECTV infection, but overexpression of Qa-1b in infected cells does. We also demonstrate that Qa-1b is down-regulated in infected and up-regulated in bystander inflammatory monocytes and B cells. Moreover, NK cells activated by ECTV infection kill Qa-1b-deficient cells in vitro and in vivo. Thus, during viral infection, recognition of Qa-1b by activating CD94/NKG2 receptors is not critical. Instead, the levels of Qa-1b expression are down-regulated in infected cells but increased in some bystander immune cells to respectively promote or inhibit their killing by activated NK cells.

Defective early innate immune response to ectromelia virus in the draining lymph nodes of aged mice due to impaired dendritic cell accumulation.

It is known that aging decreases natural resistance to viral diseases due to dysfunctional innate and adaptive immune responses, but the nature of these dysfunctions, particularly in regard to innate immunity, is not well understood. We have previously shown that C57BL/6J (B6) mice lose their natural resistance to footpad infection with ectromelia virus (ECTV) due to impaired maturation and recruitment of natural killer (NK) cells to the draining popliteal lymph node (dLN). More recently, we have also shown that in young B6 mice infected with ECTV, the recruitment of NK cells is dependent on a complex cascade whereby migratory dendritic cells (mDCs) traffic from the skin to the dLN, where they produce CCL2 and CCL7 to recruit inflammatory monocytes (iMOs). In the dLN, mDCs also upregulate NKG2D ligands to induce interferon gamma (IFN-γ) expression by group 1 innate lymphoid cells (G1-ILCs), mostly NK in cells but also some ILC1. In response to the IFN-γ, the incoming uninfected iMOs secret CXCL9 to recruit the critical NK cells. Here, we show that in aged B6 mice, the trafficking of mDCs to the dLN in response to ECTV is decreased, resulting in impaired IFN-γ expression by G1-ILCs, reduced accumulation of iMOs, and attenuated CXCL9 production by iMOs, which likely contributes to decrease in NK cell recruitment. Together, these data indicate that defects in the mDC response to viral infection during aging result in a reduced innate immune response in the dLN and contribute to increased susceptibility to viral disease in the aged.

Cutting Edge: Protection by Antiviral Memory CD8 T Cells Requires Rapidly Produced Antigen in Large Amounts.

Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design.

Zebrafish B Cell Development without a Pre-B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes.

CD79a and CD79b proteins associate with Ig receptors as integral signaling components of the B cell Ag receptor complex. To study B cell development in zebrafish, we isolated orthologs of these genes and performed in situ hybridization, finding that their expression colocalized with IgH-µ in the kidney, which is the site of B cell development. CD79 transgenic lines were made by linking the promoter and upstream regulatory segments of CD79a and CD79b to enhanced GFP to identify B cells, as demonstrated by PCR analysis of IgH-µ expression in sorted cells. We crossed these CD79-GFP lines to a recombination activating gene (Rag)2:mCherry transgenic line to identify B cell development stages in kidney marrow. Initiation of CD79:GFP expression in Rag2:mCherry+ cells and the timing of Ig H and L chain expression revealed simultaneous expression of both IgH-µ- and IgL-κ-chains, without progressing through the stage of IgH-µ-chain alone. Rag2:mCherry+ cells without CD79:GFP showed the highest Rag1 and Rag2 mRNAs compared with CD79a and CD79b:GFP+ B cells, which showed strongly reduced Rag mRNAs. Thus, B cell development in zebrafish does not go through a Raghi CD79+IgH-µ+ pre-B cell stage, different from mammals. After the generation of CD79:GFP+ B cells, decreased CD79 expression occurred upon differentiation to Ig secretion, as detected by alteration from membrane to secreted IgH-µ exon usage, similar to in mammals. This confirmed a conserved role for CD79 in B cell development and differentiation, without the requirement of a pre-B cell stage in zebrafish.

A developmental switch between fetal and adult B lymphopoiesis.

Fluorescence-activated cell sorting (FACS)-purified pro-B cells from fetal liver and adult bone marrow generate B cells with distinct phenotypes: fetal cells generate few IgD(high) B cells and half express CD5, whereas adult cells generate mostly IgD(high) cells and few express CD5. These results led us to propose a model of a developmental switch in B lymphopoiesis, similar to the well-known switch in fetal to adult erythropoiesis. More recent global analysis of mRNA and microRNA expression comparing these two types of pro-B cells revealed differential expression of Lin28b and microRNAs from the Let-7 family, indicating that this regulatory axis plays a role in the switch. Further analysis has provided data supporting this model, implicating Arid3a as a key transcription factor in mediating fetal-type B cell development. Function of this regulatory axis in human B lineage precursors may also explain the predominance of CD5(+) B cells in cord blood. We suggest that Lin28b-promoted B cell development generates many cells expressing CD5 as a consequence of positively selected self-reactivity. While such cells serve a useful role in clearance of senescent cells and in certain immune responses, they also carry the risk of progression to leukemia/lymphoma later in life.

Lin28b promotes fetal B lymphopoiesis through the transcription factor Arid3a.

Mouse B cell precursors from fetal liver and adult bone marrow (BM) generate distinctive B cell progeny when transplanted into immunodeficient recipients, supporting a two-pathway model for B lymphopoiesis, fetal "B-1" and adult "B-2." Recently, Lin28b was shown to be important for the switch between fetal and adult pathways; however, neither the mechanism of Lin28b action nor the importance of B cell antigen receptor (BCR) signaling in this process was addressed. Here, we report key advances in our understanding of the regulation of B-1/B-2 development. First, modulation of Let-7 in fetal pro-B cells is sufficient to alter fetal B-1 development to produce B cells resembling the progeny of adult B-2 development. Second, intact BCR signaling is required for the generation of B1a B cells from Lin28b-transduced BM progenitors, supporting a requirement for ligand-dependent selection, as is the case for normal B1a B cells. Third, the VH repertoire of Lin28b-induced BM B1a B cells differs from that of normal B1a, suggesting persisting differences from fetal progenitors. Finally, we identify the Arid3a transcription factor as a key target of Let-7, whose ectopic expression is sufficient to induce B-1 development in adult pro-B cells and whose silencing by knockdown blocks B-1 development in fetal pro-B cells.

Autoreactive B-1 B cells: constraints on natural autoantibody B cell antigen receptors.

B-1 B-cells constitute a distinctive population of cells that are enriched for self-reactive B cell receptors (BCRs). These BCRs are encoded by a restricted set of heavy and light chains, including heavy chains that lack nontemplated nucleotide additions at the V-D and D-J joining regions. One prototype natural autoantibody produced by B-1 B cells binds to a cryptic determinant exposed on senescent red blood cells that includes the phosphatidylcholine (PtC) moiety. The V(H)11Vkappa9 BCR, which accounts for a large fraction of the anti-PtC specificity, is underrepresented in other B-cell populations, including newly formed B cells in bone marrow, and the transitional B cells, follicular B cells, and marginal zone B cells in spleen. Previous work has shown that V(H)11 heavy chains pair ineffectively with surrogate light chain (SLC) and so do not promote development in bone marrow, but instead allow fetal liver maturation because of a fetal preference for weaker pre-BCR signaling. Such inefficient SLC pairing constitutes one constraint on the maturation of B cells containing V(H)11 rearrangements that biases their generation to fetal development. Here, we examine another possible bottleneck to the B1 cell repertoire: light chain pairing with V(H)11 heavy chain, finding very significant preferences.

Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites.

The heterochronic gene lin-28 is a regulator of developmental timing in the nematode Caenorhabditis elegans. It must be expressed in the first larval stage and downregulated by the second stage for normal development. This downregulation is mediated in part by lin-4, a 21-nt microRNA. If downregulation fails due to a mutation in a short sequence in the lin-28 3' UTR that is complementary to lin-4, then a variety of somatic cell lineages fail to progress normally in development. Here, we report that Lin-28 homologues exist in diverse animals, including Drosophila, Xenopus, mouse, and human. These homologues are characterized by the LIN-28 protein's unusual pairing of RNA-binding motifs: a cold shock domain (CSD) and a pair of retroviral-type CCHC zinc knuckles. Conservation of LIN-28 proteins shows them to be distinct from the other conserved family of CSD-containing proteins of animals, the Y-box proteins. Importantly, the LIN-28 proteins of Drosophila, Xenopus, and mouse each appear to be expressed and downregulated during development, consistent with a conserved role for this regulator of developmental timing. In addition, the extremely long 3' UTRs of mouse and human Lin-28 genes show extensive regions of sequence identity that contain sites complementary to the mammalian homologues of C. elegans lin-4 and let-7 microRNAs, suggesting that microRNA regulation is a conserved feature of the Lin-28 gene in diverse animals.

Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation.

The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.