MicroRNA processing without Dicer

The canonical processing of precursor microRNAs requires the endonuclease Dicer. A recent study shows that microRNAs can be processed independently of Dicer but instead require Argonaute 2

miRA: adaptable novel miRNA identification in plants using small RNA sequencing data

BACKGROUND: MicroRNAs (miRNAs) are short regulatory RNAs derived from longer precursor RNAs. miRNA biogenesis has been studied in animals and plants, recently elucidating more complex aspects, such as non-conserved, species-specific, and heterogeneous miRNA precursor populations. Small RNA sequencing data can help in computationally identifying genomic loci of miRNA precursors. The challenge is to predict a valid miRNA precursor from inhomogeneous read coverage from a complex RNA library: while the mature miRNA typically produces many sequence reads, the remaining part of the precursor is covered very sparsely. As recent results suggest, alternative miRNA biogenesis pathways may lead to a more diverse miRNA precursor population than previously assumed. In plants, the latter manifests itself in e.g. complex secondary structures and expression from multiple loci within precursors. Current miRNA identification algorithms often depend on already existing gene annotation, and/or make use of specific miRNA precursor features such as precursor lengths, secondary structures etc. Consequently and in view of the emerging new understanding of a more complex miRNA biogenesis in plants, current tools may fail to characterise organism-specific and heterogeneous miRNA populations. RESULTS: miRA is a new tool to identify miRNA precursors in plants, allowing for heterogeneous and complex precursor populations. miRA requires small RNA sequencing data and a corresponding reference genome, and evaluates precursor secondary structures and precursor processing accuracy; key parameters can be adapted based on the specific organism under investigation. We show that miRA outperforms the currently best plant miRNA prediction tools both in sensitivity and specificity, for data involving Arabidopsis thaliana and the Volvocine algae Chlamydomonas reinhardtii; the latter organism has been shown to exhibit a heterogeneous and complex precursor population with little cross-species miRNA sequence conservation, and therefore constitutes an ideal model organism. Furthermore we identify novel miRNAs in the Chlamydomonas-related organism Volvox carteri. CONCLUSIONS: We propose miRA, a new plant miRNA identification tool that is well adapted to complex precursor populations. miRA is particularly suited for organisms with no existing miRNA annotation, or without a known related organism with well characterized miRNAs. Moreover, miRA has proven its ability to identify species-specific miRNAs. miRA is flexible in its parameter settings, and produces user-friendly output files in various formats (pdf, csv, genome-browser-suitable annotation files, etc.). It is freely available at https://github.com/mhuttner/miRA .The authors acknowledge funding from the Deutsche Forschungsgemeinschaft (SFB 960), the Bavarian Genome Research Network (BayGene), and the Bavarian Biosystems Network (BioSysNet)

Gene silencing pathways found in the green alga Volvox carteri reveal insights into evolution and origins of small RNA systems in plants

Background: Volvox carteri (V. carteri) is a multicellular green alga used as model system for the evolution of multicellularity. So far, the contribution of small RNA pathways to these phenomena is not understood. Thus, we have sequenced V. carteri Argonaute 3 (VcAGO3)-associated small RNAs from different developmental stages. Results: Using this functional approach, we define the Volvox microRNA (miRNA) repertoire and show that miRNAs are not conserved in the closely related unicellular alga Chlamydomonas reinhardtii. Furthermore, we find that miRNAs are differentially expressed during different life stages of V. carteri. In addition to miRNAs, transposon-associated small RNAs or phased siRNA loci, which are common in higher land plants, are highly abundant in Volvox as well. Transposons not only give rise to miRNAs and other small RNAs, they are also targets of small RNAs. Conclusion: Our analyses reveal a surprisingly complex small RNA network in Volvox as elaborate as in higher land plants. At least the identified VcAGO3-associated miRNAs are not conserved in C. reinhardtii suggesting fast evolution of small RNA systems. Thus, distinct small RNAs may contribute to multicellularity and also division of labor in reproductive and somatic cells

DGK and DZHK position paper on genome editing: basic science applications and future perspective

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems

Immobility-associated thromboprotection is conserved across mammalian species from bear to human

Venous thromboembolism (VTE) comprising deep venous thrombosis and pulmonary embolism is a major cause of morbidity and mortality. Short-term immobility-related conditions are a major risk factor for the development of VTE. Paradoxically, long-term immobilized free-ranging hibernating brown bears and paralyzed spinal cord injury (SCI) patients are protected from VTE. Here we aimed to identify mechanisms of immobility-associated VTE protection in a cross-species approach. Mass spectrometry-based proteomics revealed an antithrombotic signature in platelets of hibernating brown bears with heat shock protein 47 (HSP47) as most substantially reduced protein. HSP47 downregulation or ablation attenuated immune cell activation and NET formation, contributing to thromboprotection in bears, SCI patients and mice. This cross-species conserved platelet signature may give rise to antithrombotic therapeutics and prognostic markers beyond immobility-associated VTE

microRNAs associated with the different human Argonaute proteins

MicroRNAs (miRNAs) are small noncoding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3′-untranslated region (3′-UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1–3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected

Analysis of small RNA-Argonaute complexes in different organisms

MicroRNAs (miRNAs) are small, non-coding RNAs of 20-24 nt in length. They are bound by members of the Argonaute protein family and play important roles in various processes including development, cellular homeostasis or response to external stimuli. Mis-regulations of miRNAs cause diseases such as cancers. In several chapters, this thesis describes miRNA profiles, miRNA and Argonaute protein characteristics as well as the de novo description of miRNAs and small RNAs in different cellular and organismal systems. The four human Argonaute (Ago) proteins are highly similar proteins. One distinct difference is the enzymatic activity of Ago2 (Meister et al., 2004), however, detailed studies on the relationship between Ago proteins have been few. This work shows that Ago2 is the most abundant Ago protein in the human cell lines HEK 293T and HeLa (Petri et al., 2011). Ago1 and Ago3 are expressed only to a minor amount, whereas Ago4 expression is hardly detectable. Re-analysis of their enzymatic activity showed that Ago2 remains the only Ago protein capable of cleaving RNA targets (Hauptmann et al., 2013). Following this lead, it could also be demonstrated that not only do Ago1 and Ago3 have difficulties in replacing the passenger strand (the strand of a small RNA duplex that usually gets degraded), but that by increasing the stability of the duplex, on-target effects of an siRNA could be elevated and off-target effects could be alleviated (Petri et al., 2011). To investigate the potential differences of the four Ago proteins with respect to their miRNA-binding properties, the distribution and the characteristics of miRNAs bound to endogenous Ago proteins were analyzed. By isolating Ago proteins using specific immunoprecipitation, cloning and sequencing of bound miRNAs, it could be shown that Ago proteins bind very similar sets of miRNAs. These miRNAs have in 10 to 20% of the cases non-templated additions of one adenosine. On the individual miRNA level, miRNAs can exist as 5’ or 3’ length variations that are called isomiRs. Investigating isomiRs, it could be shown that Ago2 binds to miRNAs with a broader isomiR spectrum than Ago1 and Ago3 (Dueck et al., 2012). The exceptional role of Ago2 is underscored in the processing of the non-canonical miR-451. Ago2 not only cleaves the precursor of miR-451 (Cheloufi et al., 2010; Cifuentes et al., 2010), but in my thesis it could also be shown that Ago2 exclusively binds miR-451 and does not exchange it with Ago1 or Ago3. Mutational studies on the miR-451 precursor highlight its unique and optimized structure, which renders its use as a backbone for Ago2-specific RNAi tools limited (Dueck et al., 2012). Following up on the analysis of individual miRNAs and their distribution among Ago proteins, global miRNA profiles under the influence of extracellular stimuli in an immunological setting were examined. Wild-type as well as miR-155-deficient macrophages and dendritic cells (DCs) were stimulated or matured with LPS, LDL, oxidized LDL (oxLDL) or enzymatically lysed LDL (eLDL). MiRNAs were cloned and sequenced and the resulting libraries were compared. Depending on the stimulus, miRNA profiles changed mildly to strongly. The use of miR-155 deficient cells proved to be a powerful tool to establish that miR-155 strongly influences miRNA expression. Examination and comparison the different global miRNA profiles highlighted that miR-155 is a master regulator of downstream miRNA expression. The green alga Volvox carteri is a model organism for the development of multicellularity. Its proteome and transcriptome is predicted to be highly similar to its closest unicellular relative, Chlamydomonas reinhardtii. Up to date, no protein(s) or mechanism(s) could be identified that explain the big difference in development and morphology between the two organisms. The small RNA repertoire of Volvox carteri and the proteins involved in their biogenesis and exertion of their function was not described when the project was started. Here, we characterized an Argonaute protein of V.carteri, AGO3, and analyzed AGO3-bound small RNAs. A miRNA identification pipeline was constructed and we identified 288 miRNAs contained in 140 miRNA families. Several of the predicted miRNAs were shown to be expressed and modified at their 3’ end, which is typical for plant miRNAs. Additionally, other small RNAs such as repeat-associated small RNAs or phased small RNAs were analyzed. Finally, no overlap of small RNA expression between Volvox and Chlamydomonas could be detected. The existence of a complete new set of small RNAs in Volvox carteri raises the question if the development of multicellularity and the division of labor between somatic and germ cells is at least partially regulated by miRNAs or small RNAs. Future studies, also on other members of the volvocine algae, need to elucidate the role of small RNAs in Volvox carteri further

A miR-155-dependent microRNA hierarchy in dendritic cell maturation and macrophage activation

MicroRNAs (miRNAs) are small RNAs that play important regulatory roles in many cellular pathways. MiRNAs associate with members of the Argonaute protein family and bind to partially complementary sequences on mRNAs and induce translational repression or mRNA decay. Using deep sequencing and Northern blotting, we characterized miRNA expression in wild type and miR-155-deficient dendritic cells (DCs) and macrophages. Analysis of different stimuli (LPS, LDL, eLDL, oxLDL) reveals a direct influence of miR-155 on the expression levels of other miRNAs. For example, miR-455 is negatively regulated in miR-155-deficient cells possibly due to inhibition of the transcription factor C/EBPbeta by miR-155. Based on our comprehensive data sets, we propose a model of hierarchical miRNA expression dominated by miR-155 in DCs and macrophages