Comparative analysis of small RNAs released by the filarial nematode Litomosoides sigmodontis in vitro and in vivo

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

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Mechanisms of nuclear content loading to exosomes

Science Advances ◽

10.1126/sciadv.aax8849 ◽

2019 ◽

Vol 5 (11) ◽

pp. eaax8849 ◽

Cited By ~ 37

Author(s):

Akira Yokoi ◽

Alejandro Villar-Prados ◽

Paul Allen Oliphint ◽

Jianhua Zhang ◽

Xingzhi Song ◽

...

Keyword(s):

Ovarian Cancer ◽

Small Rnas ◽

Genomic Dna ◽

Multivesicular Body ◽

Imaging Flow Cytometry ◽

Intracellular Compartments ◽

The Relationship ◽

Nuclear Content

Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development.

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In vivo and in vitro Flow Cytometry Comparative Analysis of Somatostatin-Positive Cells in the Pineal Gland of the Neonatal Rat

Neuroendocrinology ◽

10.1159/000126992 ◽

1995 ◽

Vol 62 (1) ◽

pp. 87-92 ◽

Cited By ~ 3

Author(s):

Mercè Viader ◽

Eugènia Mato ◽

Dolors Tugues ◽

Oscar Fornas ◽

Manel Puig-Domingo ◽

...

Keyword(s):

Flow Cytometry ◽

Comparative Analysis ◽

Pineal Gland ◽

Neonatal Rat

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EBV-Encoded Mirnas Facilitate Inflammation and Tumor Formation Through Both Intra- and Inter-Cellular Regulation In Vivo Mouse Model

Blood ◽

10.1182/blood.v122.21.3735.3735 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3735-3735

Author(s):

Natsuko Yamakawa ◽

Jun Ogata ◽

Takashi Yahata ◽

Jun Lu ◽

Kazuaki Yokoyama ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cells ◽

Small Rnas ◽

Conflicts Of Interest ◽

Functional Characterization ◽

Tumor Formation ◽

Sequencing Analysis ◽

Immunodeficient Mice

Abstract Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

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