Jose AM (2015) Genesis "Movement of regulatory RNA between animal cells."

Jose AM

[Genesis, 2015]

Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions.

Jose AM (2024) Elife "Heritable epigenetic changes are constrained by the dynamics of regulatory architectures."

Jose AM

[Elife, 2024]

Interacting molecules create regulatory architectures that can persist despite turnover of molecules. Although epigenetic changes occur within the context of such architectures, there is limited understanding of how they can influence the heritability of changes. Here, I develop criteria for the heritability of regulatory architectures and use quantitative simulations of interacting regulators parsed as entities, their sensors, and the sensed properties to analyze how architectures influence heritable epigenetic changes. Information contained in regulatory architectures grows rapidly with the number of interacting molecules and its transmission requires positive feedback loops. While these architectures can recover after many epigenetic perturbations, some resulting changes can become permanently heritable. Architectures that are otherwise unstable can become heritable through periodic interactions with external regulators, which suggests that mortal somatic lineages with cells that reproducibly interact with the immortal germ lineage could make a wider variety of architectures heritable. Differential inhibition of the positive feedback loops that transmit regulatory architectures across generations can explain the gene-specific differences in heritable RNA silencing observed in the nematode <i>Caenorhabditis elegans</i>. More broadly, these results provide a foundation for analyzing the inheritance of epigenetic changes within the context of the regulatory architectures implemented using diverse molecules in different living systems.

Jose AM (2020) Elife "The analysis of living systems can generate both knowledge and illusions."

Jose AM

[Elife, 2020]

Life relies on phenomena that range from changes in molecules that occur within nanoseconds to changes in populations that occur over millions of years. Researchers have developed a vast range of experimental techniques to analyze living systems, but a given technique usually only works over a limited range of length or time scales. Therefore, gaining a full understanding of a living system usually requires the integration of information obtained at multiple different scales by two or more techniques. This approach has undoubtedly led to a much better understanding of living systems but, equally, the staggering complexity of these systems, the sophistication and limitations of the techniques available in modern biology, and the need to use two or more techniques, can lead to persistent illusions of knowledge. Here, in an effort to make better use of the experimental techniques we have at our disposal, I propose a broad classification of techniques into six complementary approaches: perturbation, visualization, substitution, characterization, reconstitution, and simulation. Such a taxonomy might also help increase the reproducibility of inferences and improve peer review.

Jose AM et al. (2007) Annu Rev Genet "Transport of sequence-specific RNA interference information between cells."

Hunter CP, Jose AM

[Annu Rev Genet, 2007]

When eukaryotic cells encounter double-stranded RNA, genes of matching sequence are silenced through RNA interference. Surprisingly, in some animals and plants, the same gene is specifically silenced even in cells that did not encounter the double-stranded RNA, due to the transport of a gene-specific silencing signal between cells. This silencing signal likely has an RNA component that gives it sequence-specificity, however its precise identity remains unknown. Studies in the worm Caenorhabditis elegans and in plants have revealed parts of a complex protein machinery that transports this silencing signal. Some of these proteins are conserved in vertebrates, including mammals, raising the possibility that higher animals can communicate gene-specific silencing information between cells. Such communication provides antiviral immunity in plants and perhaps in C. elegans. Identifying the transported silencing signal and deciphering the evolutionarily selected role of the transport machinery are some of the key challenges for the future. Expected final online publication date for the Annual Review of Genetics Volume 41 is November 10, 2007. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Jose AM et al. (2011) Nat Struct Mol Biol "Two classes of silencing RNAs move between Caenorhabditis elegans tissues."

Garcia GA, Hunter CP, Jose AM

[Nat Struct Mol Biol, 2011]

Organism-wide RNA interference (RNAi) is due to the transport of mobile silencing RNA throughout the organism, but the identities of these mobile RNA species in animals are unknown. Here, we present genetic evidence that both the initial double-stranded RNA (dsRNA), which triggers RNAi, and at least one dsRNA intermediate produced during RNAi can act as or generate mobile silencing RNA in C. elegans. This dsRNA intermediate requires the long dsRNA-binding protein RDE-4, the endonuclease DCR-1, which cleaves long dsRNA into double-stranded short-interfering RNA (ds-siRNA), and the putative nucleotidyltransferase MUT-2 (RDE-3). However, single-stranded siRNA and downstream secondary siRNA produced upon amplification by the RNA-dependent RNA polymerase RRF-1 do not generate mobile silencing RNA. Restricting intertissue transport to long dsRNA and directly processed siRNA intermediates rather than amplified siRNA may serve to modulate the extent of systemic silencing in proportion to available dsRNA.

Jose AM et al. (2012) Proc Natl Acad Sci U S A "Conserved tyrosine kinase promotes the import of silencing RNA into Caenorhabditis elegans cells."

Leal-Ekman S, Kim YA, Jose AM, Hunter CP

[Proc Natl Acad Sci U S A, 2012]

RNA silencing in Caenorhabditis elegans is transmitted between cells by the transport of double-stranded RNA (dsRNA). The efficiency of such transmission, however, depends on both the cell type and the environment. Here, we identify systemic RNAi defective-3 (SID-3) as a conserved tyrosine kinase required for the efficient import of dsRNA. Without SID-3, cells perform RNA silencing well but import dsRNA poorly. Upon overexpression of SID-3, cells import dsRNA more efficiently than do wild-type cells and such efficient import of dsRNA requires an intact SID-3 kinase domain. The mammalian homolog of SID-3, activated cdc-42-associated kinase (ACK), acts in many signaling pathways that respond to environmental changes and is known to directly associate with endocytic vesicles, which have been implicated in dsRNA transport. Therefore, our results suggest that the SID-3/ACK tyrosine kinase acts as a regulator of RNA import into animal cells.

Jose AM et al. (2009) Proc Natl Acad Sci U S A "Export of RNA silencing from C. elegans tissues does not require the RNA channel SID-1."

Hunter CP, Smith JJ, Jose AM

[Proc Natl Acad Sci U S A, 2009]

Double-stranded RNA (dsRNA) triggers RNA interference (RNAi) to silence genes of matching sequence. In some animals this experimentally induced silencing is transported between cells, and studies in the nematode Caenorhabditis elegans have shown that the dsRNA channel SID-1 is required for the import of such transported silencing signals. Gene silencing can also be triggered by endogenously expressed RNAi triggers, but it is unknown whether such silencing is transported between cells. Here, we show that, in C. elegans, SID-1 is required for efficient silencing of multicopy transgenes, indicating that mobile silencing signals contribute to transgene silencing. Further, most tissues can transport silencing initiated by the tissue-specific transgenic expression of RNAi triggers to other tissues, consistent with expressed RNAi triggers generating mobile silencing signals. Whereas the import of silencing signals requires SID-1, we found that mobile silencing signals generated by transgene-expressed RNAi triggers are exported to other tissues through a SID-1-independent mechanism. Furthermore, when RNAi triggers are expressed in ingested Escherichia coli, silencing signals can be transported to internal tissues from the gut lumen across gut cells that lack SID-1. Thus, C. elegans can transport endogenous and exogenous RNA silencing signals between many different tissues via at least 2 SID-1 independent export pathways.

Jose AM et al. (2005) J Biol Chem "Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo."

Jose AM, Koelle MR

[J Biol Chem, 2005]

Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the C. elegans ortholog of human neuronal DGKtheta;, inhibits neurotransmission and thus controls behavior. DGK-1, like DGKtheta;, has three cysteine-rich domains (CRDs), a pleckstrin homology (PH) domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1 controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are either in the second CRD, the third CRD, or in the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact PH domain yet appear to have significant function. Additional novel isoform(s) account for all the DGK-1 function necessary for one behavior, dopamine response.

Jose AM et al. (2007) Genetics "A specific subset of transient receptor potential vanilloid-type channel subunits in Caenorhabditis elegans endocrine cells function as mixed heteromers to promote neurotransmitter release."

Jose AM, Koelle MR, Bany IA, Chase DL

[Genetics, 2007]

Transient receptor potential (TRP) channel subunits form homotetramers that function in sensory transduction. Heteromeric channels also form, but their physiological subunit compositions and functions are largely unknown. We found a dominant-negative mutant of the C. elegans TRPV (vanilloid-type) subunit OCR-2 that apparently incorporates into and inactivates OCR-2 homomers as well as heteromers with the TRPV subunits OCR-1 and -4, resulting in a premature egg-laying defect. This defect is reproduced by knocking out all three OCR genes, but not by any single knockout. Thus a mixture of redundant heteromeric channels prevents premature egg laying. These channels, as well as the G protein Galphao, function in neuroendocrine cells to promote release of neurotransmitters that block egg laying until eggs filling the uterus deform the neuroendocrine cells. The TRPV channel OSM-9, previously suggested to be an obligate heteromeric partner of OCR-2 in sensory neurons, is expressed in the neuroendocrine cells but has no detectable role in egg laying. Our results identify a specific set of heteromeric TRPV channels that redundantly regulate neuroendocrine function, and show a subunit combination that functions in sensory neurons is also present in neuroendocrine cells but has no detectable function in these cells.

Saenz-Narciso, Beatriz et al. (2011) International Worm Meeting "Characterizing the adrenomedullin homologue in C. elegans."

Saenz-Narciso, Beatriz, Gomez-Orte, Eva, Martinez, Alfredo, Cabello, Juan

[International Worm Meeting, 2011]

Adrenomedullin (AM) is a peptide hormone that shows multiple physiological functions in vertebrates, such as bronchodilation, neurotransmission, hormone regulation, antimicrobial activity or growth regulation. Deregulation of AM has been shown in different human pathologies such as diabetes, cancer, hypertension, heart failure, and sepsis. Research on molecules capable of modulating AM and its effects may lead to the discovery of new pharmacological agents to treat these important human diseases. Highly interesting is the role of AM in cancer. It has been proven that AM acts as an autocrine/paracrine tumor cell survival factor and its elevated expression in cancer cells can promote angiogenesis leading to tumor progression. While AM has been well characterized in other research models, little is known about this molecule in C. elegans. We have found and characterized a mutant in the AM homologue of C.elegans, here called wam-1. 4D microscopy analysis of wam-1 mutants shows defects in the migration of the hermaphrodite gonad. There are also several defects in embryonic development. Some embryos have cells that are excluded out of the worm during development while fate specification, as well as proliferation is normal. This suggests a function of wam-1 in cell adhesion. To localize the expression of the worm AM homologue protein, we performed inmunostaining using mouse anti-AM antibodies. The AM protein was localized in the hypodermis. In contrast, we also generated transgenic lines expressing GFP under the control of the wam-1 promoter. GFP expression was detected in the mouth of the worm, although due to its homology with secreted hormones, the protein could play a role far away from the secretory cells. At the moment, we are performing a detailed molecular and genetic characterization, in order to understand the function of this molecule.