DDX24, a D-E-A-D box RNA helicase, is required for muscle fiber organization and anterior pole specification essential for head regeneration in planarians

ABSTRACTPlanarians have a remarkable ability to undergo whole-body regeneration. The timely establishment of polarity at the wound site followed by the specification of the organizing centers- the anterior pole and the posterior pole, are indispensable for successful regeneration. In planarians, polarity, pole, and positional-information determinants are predominantly expressed by muscles. The molecular toolkit that enables this functionality of planarian muscles however remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase and the homolog of human DDX24, is critical for planarian head regeneration. DDX24 is enriched in muscles and its knockdown leads to defective muscle-fiber organization and failure to re-specify anterior pole/organizer. Overall, loss of DDX24 manifests into gross misregulation of many well-characterized positional-control genes and patterning-control genes, necessary for organogenesis and tissue positioning and tissue patterning. In addition, wound-induced Wnt signalling was also upregulated in ddx24 RNAi animals. Canonical WNT-βCATENIN signalling is known to suppress head identity throughout bilateria, including planarians. Modulating this Wnt activity by β-catenin-1 RNAi, the effector molecule of this pathway, partially rescues the ddx24 RNAi phenotype, implying that a high Wnt environment in ddx24 knockdown animals likely impedes their normal head regeneration. Furthermore, at a sub-cellular level, RNA helicases are known to regulate muscle mass and function by regulating their translational landscape. ddx24 knockdown leads to the downregulation of large subunit ribosomal RNA and the 80S ribosome peak, implying its role in ribosome biogenesis and thereby influencing the translational output. This aspect seems to be an evolutionarily conserved role of DDX24. In summary, our work demonstrates the role of a D-E-A-D box RNA helicase in whole-body regeneration through muscle fiber organization, and pole and positional-information re-specification, likely mediated through translation regulation.

Genomic analyses reveal FoxG as an upstream regulator of wnt1 required for posterior identity specification in planarians

Embryonic specification of the first body axis requires the formation of an Organizer, a group of cells with the ability to instruct fates in the surrounding tissue. The existence of organizing regions in adults, i.e. during regeneration, which also requires patterning of new tissues, remains unstudied. To that aim, we study regeneration in planarians, flatworms that can regenerate any missing structure, even the head, in a few days. In planarians, as described in embryonic models, the cWNT pathway specifies the anterior-posterior axis. During the first 12-24h after amputation both wnt1 and notum (a Wnt inhibitor) are expressed in any wound, but 48 hours later they become restricted to posterior or anterior facing wounds, forming the anterior and the posterior organizers, respectively. In this study we undertook a genomic approach to further understand the mechanism that triggers the early expression of wnt1 and the specification of the posterior identity. Through ATAC-sequencing and CHIPmentation techniques we uncovered Cis-Regulatory Elements of Schmidtea mediterranea genome and analyzed them in notum and wnt1 (RNAi) animals. The result shows that already at 12 hours after amputation the chromatin structure of the wounds has changed its conformation according to the polarity of the pre-existing tissue. Analysing the DNA binding motives present in the proximal regulatory regions of genes down-regulated after wnt1 (RNAi) we found a few genes containing a TCF binding site, which include posterior Homeobox genes and chromatin remodelling proteins, suggesting that those are direct targets of the cWNT pathway and the responsible to trigger the expression of the posterior effectors. Furthermore, we have identified FoxG as an up-stream regulator of wnt1 transcription, probably though binding to an enhancer found in its first intron. Silencing of foxG inhibits the early phase of wnt1 expression and phenocopies the wnt1 (RNAi) phenotype, indicating its early role in specifying posterior versus anterior identity. Moreover, we have created a new open platform to interpret all transcriptomic and genomic results obtained (https://compgen.bio.ub.edu/PlanNET/planexp).

Moonlighting α-PheRS connects JAK/STAT with Notch signaling to modulate intestinal homeostasis

Summary / AbstractAminoacyl tRNA synthetases (aaRSs) not only load the appropriate amino acid onto their cognate tRNA, many of them perform additional functions that are not necessarily related to their canonical activities. Phenylalanyl tRNA synthetase (PheRS/FARS) levels are elevated in multiple cancers and, interestingly, also in normal stem cells. Our results show that elevated expression of the α-PheRS subunit stimulates cell proliferation in different tissues, while downregulation of α-PheRS reduces organ size. The stimulation of proliferation is independent of the β-PheRS subunit and the aminoacylation activity, and it does not visibly stimulate translation. Furthermore, stem cell-specific overexpression of α-PheRS caused a tumor phenotype in the intestine, a phenotype indistinguishable from the Notch RNAi phenotype in the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling whether Notch induces differentiation into enterocytes or neuroblast stem cell proliferation. Indeed, in the wing discs system, elevated α-PheRS levels suppress Notch transcriptional activity. α-PheRS therefore seems to act as a general regulator of Notch signaling, and its own levels are in turn controlled by Stat92E, the transcription factor of the JAK/STAT signaling pathway that is needed for the differentiation of intestinal stem cells during tissue homeostasis under normal conditions. From this we conclude that the α-PheRS subunit can transmit the activity status of the JAK/STAT pathway to the Notch pathway as a mechanism to coordinate stem cell proliferation with differentiation. In this process, α-PheRS levels balance between tissue development and tissue growth to regulate tissue homeostasis.

Structure-function analysis reveals a DNA polymerization-independent role for mitochondrial DNA polymerase IC in African trypanosomes

ABSTRACTThe mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles called kinetoplast DNA (kDNA). Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.Summary statementMitochondrial DNA segregation in African trypanosomes is supported by a dual-functioning DNA polymerase.

Functional Interaction between Apolipophorins and Complement Regulate the Mosquito Immune Response to Systemic Infections

The complement-like protein thioester-containing protein 1 (TEP1) is the hallmark effector molecule against Plasmodium ookinetes in the malaria vector Anopheles gambiae. We have previously shown that the knockdown of the noncatalytic clip domain serine protease CLIPA2 increased TEP1-mediated killing rendering mosquitoes more resistant to Plasmodium, bacterial and fungal infections. Here, CLIPA2 coimmunoprecipitation from the hemolymph of Beauveria bassiana-infected mosquitoes followed by mass spectrometry and functional genetic analysis led to the identification of the Apolipophorin-II/I gene, encoding the two lipid carrier proteins Apo-I and II, as a novel negative regulator of TEP1-mediated immune response during mosquito systemic infections. Apo-II/I exhibits a similar RNAi phenotype as CLIPA2 in mosquito bioassays characterized by increased resistance to B. bassiana and Escherichia coli infections. We provide evidence that this enhanced resistance to systemic infections is TEP1 dependent. Interestingly, silencing Apo-II/I but not CLIPA2 upregulated the expression of TEP1 following systemic infections with E. coli and B. bassiana in a c-Jun N-terminal kinase pathway-dependent manner. Our results suggest that mosquito Apo-II/I plays an important immune regulatory role during systemic infections and provide novel insight into the functional interplay between lipid metabolism and immune gene regulation.

RNA interference and protein expression of the four calmodulin-like genes in Caenorhabditis elegans

Calmodulin (CaM) is a major intracellular calcium receptor and probably the best studied member of the EF hand family of eukaryotic calcium-binding proteins. In contrast, much less is known about the related CaM-like proteins in animals and plants. We have previously characterized an embryonic phenotype of the single CaM gene cmd-1 from the nematode Caenorhabditis elegans. A previous functional high throughput study in C. elegans reported an embryonic lethal RNAi phenotype also for the related CaM-like gene cal-2. Based on these results we aimed to analyse the reported embryonic lethal cal-2 RNAi phenotype and compare it to that of the CaM cmd-1 embryo. Unfortunately, we were not able to find any visible RNAi phenotype for cal-2 as well as for the three remaining nematode cal genes cal-1, cal-3 and cal-4. However, because CaMs often have long cellular half-lives, we think that a definitive picture about the functionality of these genes will be established once corresponding mutant strains will be available. Immunofluorescence analyses revealed a significant expression of the CAL-2 and CAL-4 proteins in the body wall muscle indicating a possible functional redundancy of both these proteins in the C. elegans muscle.

RNA interference in plant parasitic nematodes: a summary of the current status

SUMMARYRNA interference (RNAi) has emerged as an invaluable gene-silencing tool for functional analysis in a wide variety of organisms, particularly the free-living model nematode Caenorhabditis elegans. An increasing number of studies have now described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when nematodes take up double stranded RNA (dsRNA) or short interfering RNAs (siRNAs) that elicit a systemic RNAi response. Despite many successful reports, there is still poor understanding of the range of factors that influence optimal gene silencing. Recent in vitro studies have highlighted significant variations in the RNAi phenotype that can occur with different dsRNA concentrations, construct size and duration of soaking. Discrepancies in methodology thwart efforts to reliably compare the efficacy of RNAi between different nematodes or target tissues. Nevertheless, RNAi has become an established experimental tool for plant parasitic nematodes and also offers the prospect of being developed into a novel control strategy when delivered from transgenic plants.

The AP-2 Adaptor β2 Appendage Scaffolds Alternate Cargo Endocytosis

The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites.

The Scaffold Protein POSH Regulates Axon Outgrowth

How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth. Complementation analysis reveals a requirement for the third Src homology (SH) 3 domain of POSH, and we find that the actomyosin regulatory protein Shroom3 interacts with this domain of POSH. Inhibition of Shroom3 expression by RNAi leads to increased process lengths, as observed for POSH RNAi, suggesting that POSH and Shroom function together to inhibit process outgrowth. Complementation analysis and interference of protein function by dominant-negative approaches suggest that Shroom3 recruits Rho kinase to inhibit process outgrowth. Furthermore, inhibition of myosin II function reverses the POSH or Shroom3 RNAi phenotype, indicating a role for myosin II regulation as a target of the POSH–Shroom complex. Collectively, these results suggest that the molecular scaffold protein POSH assembles an inhibitory complex that links to the actin–myosin network to regulate neuronal process outgrowth.