Non-canonical RNA substrates of Drosha lack many of the conserved features found in primary microRNA stem-loops.

The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it is required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and appears to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha have yet to be characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs) and compared the features of non-canonical versus miRNA stem-loop substrates. mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these mRNA substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNA stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential.

The αß and γδ T cell lineages both differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as CD4-CD8- double negative 1 (DN1), which has previously been shown to be a heterogenous mixture of cells. Of these, only the CD117+ fraction has been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αß and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells may be derived from a subset of CD117- DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed a single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages indeed comprise a transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell responses, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell responses.

MYL9 deficiency is neonatal lethal in mice due to abnormalities in the lung and the muscularis propria of the bladder and intestine.

Class II myosin complexes are responsible for muscle contraction as well as other non-sarcomeric contractile functions in cells. Myosin heavy chain molecules form the core of these structures, while light chain molecules regulate their stability and function. MYL9 is a light chain isoform that is thought to regulate non-sarcomeric myosin. However, whether this in only in specific cell types or in all cells remains unclear. To address this, we generated MYL9 deficient mice. These mice die soon after birth with abnormalities in multiple organs. All mice exhibited a distended bladder, shortening of the small intestine and alveolar overdistension in the lung. The Myl9 allele in these mice included a LacZ reporter knockin that allowed for mapping of Myl9 gene expression. Using this reporter, we show that MYL9 expression is restricted to the muscularis propria of the small intestine and bladder, as well as in the smooth muscle layer of the bronchi in the lung and major bladder vessels in all organs. This suggests that MYL9 is important for the function of smooth muscle cells in these organs. Smooth muscle dysfunction is therefore likely to be the cause of the abnormalities observed in the intestine, bladder and lung of MYL9 deficient mice and the resulting neonatal lethality.

Inhibition of the antigen-presenting ability of dendritic cells by non-structural protein 2 of influenza A virus.

Influenza A viruses (IAV), including human IAV and avian IAV (H9N2 subtype), are recurring of influenza outbreaks worldwide in a wide range of mammalian and avian species. Dendritic cells (DCs) are specialised antigen presenting cells. Although DCs can take up IAV and transmit it to other cells, it still unclear why DCs do not effectively present IAV antigens. In this study, we found that Non-structural protein 2 (NS2) of IAV inhibited the maturation and antigen-presenting ability of DCs. We then examined a potential involvement of microRNAs (miRNAs). Analyses of avian DCs stimulated with avian IAV identified 9 upregulated and 10 downregulated miRNAs. However, nearly none microRNA has been significantly altered by NS2 stimulation. Moreover, we found that NS2 binds to exportin 5 (Xpo5), which inhibited miRNA biogenesis. Thus, hijacking of the miRNA biogenesis pathway appears to be one mechanism by which NS2 impairs antigen presentation. Furthermore, we found that NS2 directly interacts with interferon regulatory factor 3, which also inhibits the antigen-presenting ability of DCs. These results thus indicate that NS2-mediated impairment of antigen presentation by DCs might be a mechanism that contributes to the prevalence of the influenza virus.

Single-Cell RNA Sequencing Approaches for Tracing T Cell Development.

T cell development occurs in the thymus, where uncommitted progenitors are directed into a range of sublineages with distinct functions. The goal is to generate a TCR repertoire diverse enough to recognize potential pathogens while remaining tolerant of self. Decades of intensive research have characterized the transcriptional programs controlling critical differentiation checkpoints at the population level. However, greater precision regarding how and when these programs orchestrate differentiation at the single-cell level is required. Single-cell RNA sequencing approaches are now being brought to bear on this question, to track the identity of cells and analyze their gene expression programs at a resolution not previously possible. In this review, we discuss recent advances in the application of these technologies that have the potential to yield unprecedented insight to T cell development.

A comparison of alternative mRNA splicing in the CD4 and CD8 T cell lineages.

T cells can be subdivided into a number of different subsets that are defined by their distinct functions. While the specialization of different T cell subsets is partly achieved by the expression of specific genes, the overall transcriptional profiles of all T cells appear very similar. Alternative mRNA splicing is a mechanism that facilitates greater transcript/protein diversity from a limited number of genes, which may contribute to the functional specialization of distinct T cell subsets. In this study we employ a combination of short-read and long-read sequencing technologies to compare alternative mRNA splicing between the CD4 and CD8 T cell lineages. While long-read technology was effective at assembling full-length alternatively spliced transcripts, the low sequencing depth did not facilitate accurate quantitation. On the other hand, short-read technology was ineffective at assembling full-length transcripts but was highly accurate for quantifying expression. We show that integrating long-read and short-read data together achieves a more complete view of transcriptomic diversity. We found that while the overall usage of transcript isoforms was very similar between the CD4 and CD8 lineages, there were numerous alternative spliced mRNA isoforms that were preferentially used by one lineage over the other. These alternative spliced isoforms included ones with different exon usage, exon exclusion or intron inclusion, all of which are expected to significantly alter the protein sequence.

Regulating gene expression in animals through RNA endonucleolytic cleavage.

The expression of any gene must be precisely controlled for appropriate function. This expression can be controlled at various levels. This includes epigenetic regulation through DNA methylation or histone modifications. At the posttranscriptional level, regulation can be via alternative splicing or controlling messenger RNA (mRNA) stability. RNA cleavage is one way to control mRNA stability. For example, microRNA (miRNA)-induced mRNA cleavage has long been recognised in plants. RNA cleavage also appears to be widespread in other kingdoms of life, and it is now clear that mRNA cleavage plays critical functions in animals. Although miRNA-induced mRNA cleavage can occur in animals, it is not a widespread mechanism. Instead, mRNA cleavage can be induced by a range of other mechanisms, including by endogenous short inhibitory RNAs (endo-siRNAs), as well as the Ribonuclease III (RNase III) enzymes Drosha and Dicer. In addition, RNA cleavage induced by endo-siRNAs and PIWI-interacting RNAs (piRNAs) is important for genome defence against transposons. Moreover, several RNase has been identified as important antiviral mediators. In this review, we will discuss these various RNA endonucleolytic cleavage mechanisms utilised by animals to regulate the expression of genes and as a defence against retrotransposons and viral infection.

Biologically active constituents of the secretome of human W8B2+ cardiac stem cells.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

Granzyme A Deficiency Breaks Immune Tolerance and Promotes Autoimmune Diabetes Through a Type I Interferon-Dependent Pathway.

Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A-deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.

miRNAs Are Essential for the Regulation of the PI3K/AKT/FOXO Pathway and Receptor Editing during B Cell Maturation.

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

MicroRNAs in CD4(+) T cell subsets are markers of disease risk and T cell dysfunction in individuals at risk for type 1 diabetes.

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4(+) FOXP3(+) T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4(+) T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4(+) T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4(+) T cell subsets are markers of risk and T cell dysfunction in T1D.

Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (ß3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband ß3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/ß3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband ß3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband ß3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.

Drosha controls dendritic cell development by cleaving messenger RNAs encoding inhibitors of myelopoiesis.

To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.

A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity.

We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis.

Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

A microRNA expression atlas of mouse dendritic cell development.

Dendritic cells (DCs) are sentinel cells of the immune system and are essential for inducing a proper immune response. The mechanisms driving the development of DCs are not fully understood. Although the roles of cytokines and transcription factors have been a major focus, there is now substantial interest in the role of microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression by targeting messenger RNAs for translational repression and ultimately degradation. By means of deep sequencing, we have assembled a comprehensive and quantitative resource of miRNA expression during DC development. We show that mature DCs and their hematopoietic progenitors can be distinguished based on miRNA expression profiles. On the other hand, we show that functionally distinct conventional and plasmacytoid DC subsets are indistinguishable based on miRNA profile. In addition, we identify differences between ex vivo purified conventional DCs and their in vitro Flt3L-generated counterparts. This miRNA expression atlas will provide a valuable resource for the study of miRNAs in DC development and function.

The role of microRNAs in lymphopoiesis.

The immune system is composed of a diverse range of cell types, each with a distinct function. It can be broadly divided into the lymphoid (T, B, NK, etc.) and myeloid (monocyte, granulocyte, etc.) arms. Lymphopoiesis, the development and differentiation of lymphoid lineages, has been studied extensively for decades. For example, the influence of extracellular signals, signaling pathways and transcription factors has already been well documented. However, the importance of microRNAs has been highlighted by a surge of studies in recent years. In this review, we will discuss what is currently known about the role of microRNAs in lymphopoiesis, from the hematopoietic stem cell through to the differentiation of mature lymphocytes including thymic development, helper and regulatory T cells, fate determination of B cells and dendritic cells.

The miR-17 ∼ 92a cluster of microRNAs is required for the fitness of Foxp3+ regulatory T cells.

By genetic inactivation of key microRNA biogenesis enzymes, we and others have previously demonstrated the critical requirement of the microRNA pathway for the differentiation and function of Foxp3(+) regulatory T cells. In this study, we identified members of the miR-17 ∼ 92a cluster of microRNAs to be enriched in regulatory T cells. To investigate the function of this microRNA cluster, we deleted the gene specifically in Foxp3(+) cells in mice. We found that miR-17 ∼ 92a is required for the fitness of regulatory T cells, and deficiency impacted at the level of apoptosis and proliferation of these cells. This led to a loss of Foxp3(+) cells over time, particularly in competitive settings, and culminated in a range of immunologic perturbations. Thus, miR-17 ∼ 92a-target interactions are part of the essential microRNA networks that safeguard the regulatory T cell lineage.

MicroRNA-independent roles of the RNase III enzymes Drosha and Dicer.

The ribonuclease III enzymes Drosha and Dicer are renowned for their central roles in the biogenesis of microRNAs (miRNAs). For many years, this has overshadowed the true versatility and importance of these enzymes in the processing of other RNA substrates. For example, Drosha also recognizes and cleaves messenger RNAs (mRNAs), and potentially ribosomal RNA. The cleavage of mRNAs occurs via recognition of secondary stem-loop structures similar to miRNA precursors, and is an important mechanism of repressing gene expression, particularly in progenitor/stem cell populations. On the other hand, Dicer also has critical roles in genome regulation and surveillance. These include the production of endogenous small interfering RNAs from many sources, and the degradation of potentially harmful short interspersed element and viral RNAs. These findings have sparked a renewed interest in these enzymes, and their diverse functions in biology.