Pal AS et al. (2017) Adv Cancer Res "Animal Models to Study MicroRNA Function."

Kasinski AL, Pal AS

[Adv Cancer Res, 2017]

The discovery of the microRNAs, lin-4 and let-7 as critical mediators of normal development in Caenorhabditis elegans and their conservation throughout evolution has spearheaded research toward identifying novel roles of microRNAs in other cellular processes. To accurately elucidate these fundamental functions, especially in the context of an intact organism, various microRNA transgenic models have been generated and evaluated. Transgenic C. elegans (worms), Drosophila melanogaster (flies), Danio rerio (zebrafish), and Mus musculus (mouse) have contributed immensely toward uncovering the roles of multiple microRNAs in cellular processes such as proliferation, differentiation, and apoptosis, pathways that are severely altered in human diseases such as cancer. The simple model organisms, C. elegans, D. melanogaster, and D. rerio, do not develop cancers but have proved to be convenient systesm in microRNA research, especially in characterizing the microRNA biogenesis machinery which is often dysregulated during human tumorigenesis. The microRNA-dependent events delineated via these simple in vivo systems have been further verified in vitro, and in more complex models of cancers, such as M. musculus. The focus of this review is to provide an overview of the important contributions made in the microRNA field using model organisms. The simple model systems provided the basis for the importance of microRNAs in normal cellular physiology, while the more complex animal systems provided evidence for the role of microRNAs dysregulation in cancers. Highlights include an overview of the various strategies used to generate transgenic organisms and a review of the use of transgenic mice for evaluating preclinical efficacy of microRNA-based cancer therapeutics.

Pal A et al. (2019) Dev Cell "Defend Thyself and Thy Offspring."

Shah VN, Pal A, Simard MJ

[Dev Cell, 2019]

Three recent studies (Dodson and Kennedy, 2019; Lev etal., 2019; Ouyang etal., 2019) reveal that germ granule formation is necessary to protect germline-expressed genes from improper small RNA-mediated silencing. Loss of this protection leads to accumulation of small RNAs, impacting gene expression in multiple subsequent generations.

Edgar LG et al. (1992) Worm Breeder's Gazette "PAL-ing Around with nob-2"

Wood WB, Carr SH, Edgar LG

[Worm Breeder's Gazette, 1992]

We are investigating two trimethylpsoralen-induced non-maternal mutations, ct224 and ct281 ,which define a complementation group temporarily termed "nob-2 "and result in hatching L1 swith a fairly normal anterior and a grossly defective (nob-like) posterior; these animals die in a day or two. The first visible defect is abnormal arrangement of the 8 E cells at approximately the 200 cell stage. The mutations map to LG III between unc-79 and dpy-17 ,and fail to complement the male ray phenotype of pal-1 (e2091),obtained from C. Kenyon(1). ct224 shows slightly more severe defects than ct281 .We have probed Southern blots with fragments from three nested genomic subclones containing pal-1 sequences (see diagram), generously provided by D. Waring and C. Kenyon, and find polymorphisms associated with both alleles. ct224 deletes approximately 4kb, removing the 3' pal-1 exon and part or all of the next exon containing the homeobox sequence ceh-3 (2).ct281 deletes approximately 1 kb, and removes only the 3' exon. [See Figure 1] Since these are deletions, we are still faced with the possibility that they disrupt a second gene lying close to or overlapping pal-1 .Northern blots using pWPK6 probes identify two transcripts of approximately 1.25 and 1.45 kb (the pal-1 cDNA is 1.3 kb and presumably full-length, since the 5' end contains part of the splice leader sequence(3)), present in RNA from eggs and mixed population worms, and at low levels in L1 RNA. An additional transcript (800 bp) is identified by probes just 3' to pWPK6 .In rescue experiments, injection of pWPK6 into ct224 unc-32 dpy-18 /qC1gave Unc Dpy F1 sthat produced only Nob progeny, indicating complementation, but we have not yet obtained a transformed line with any of the plasmids or the cosmid WO5E6 ,which covers this region. Although more experiments will be required to rule out the possibility that the nob-2 mutations affect an overlapping gene, these results suggest that they are pal-1 null mutations. Thus there may be an embryonic posterior patterning function for this gene, which postembryonically mediates cell communication in the male tail, possibly by regulating the homeobox gene mab-5 (1,3).The pal-1 homeobox has similarities to the Drosophila gene caudal and the mouse gene cdx-1 ,both of which also appear to be involved in organization of posterior embryonic endoderm.

Edgar LG et al. (1991) International C. elegans Meeting "'NOBS' AND AN EMBRYONIC ROLE FOR PAL- 1"

Wood WB, Notka F, Wolf N, Edgar LG

[International C. elegans Meeting, 1991]

The embryonic lethal phenotypes ('no back end') resulting from psoralen-induced non-maternal mutations at two loci suggest roles for these genes in posterior embryonic morphogenesis. The 'nob-l' locus maps between spe-6 and unc-25 on the right arm of LGIII. The ct223 allele is a neonatal lethal with a spherical gut and a round posterior, but a normal head; its earliest visible defect is a misarrangement of the E cells in the 100-cell embryo. ct230 is a weaker homozygous viable allele, (approximately 65% fertility) which results in a lumpy deformed tail very similar to vab-7. ct230/eDf2 is nonviable, suggesting that ct223 is a hypomorph, while ct223/eDf2 has the same phenotypic range as ct223/ct223. We have also isolated a larger deficiency of the locus with a break point between spe-6 and 'nob-l' extending at least to bli-S. We are mapping Tcl polymorphisms in this region as an approach to cloning the gene. ct224, which results in a similar but slightly more severe posterior embryonic defect, was isolated in a LGIII screen for more alleles of 'nob-l'. However, it complements 'nob' alleles, maps between dpy17 and unc-79 and fails to complement pal-l, the homeobox gene identified in Cynthia Kenyon's lab, which is involved in postembryonic cell interactions of V6 and T in the male tail. Because ct224 is psoralen-induced and might be a deletion of more than one gene, we are trying to rescue the mutant phenotype by injecting the pal-l plasmids, given us by Cynthia ( thanks!). So far we have no established lines, but we have seen transient rescue in several injection series, including those using the smallest plasmid pWPK6 (5.5 kb). At present we believe that ct224 is a strong, probably a null, allele of pal1, indicating an embryonic role for this gene. We are beginning mosaic analysis to determine which embryonic lineages require the gene function. The double mutant ct223ct224 has a somewhat additive phenotype (hatched embryos look like pears). Construction of double mutants with the weaker phenotypes is planned, and may be more informative as to gene interactions. It is of interest that the homeobox of pal-l shows sequence similarity to that of the caudal gene in Drosophila and its apparent mouse homolog, which also are implicated in posterior/endodermal development.

Pal A et al. (2024) Nucleic Acids Res "Defining the contribution of microRNA-specific Argonautes with slicer capability in animals."

Lantin M, Maniates KA, Simard MJ, Vasudevan V, Pal A, Abbott AL, Houle F, Huberdeau MQ

[Nucleic Acids Res, 2024]

microRNAs regulate gene expression through interaction with an Argonaute protein. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicer residues in the canonical microRNA pathway is still unclear in animals. To address this, we created Caenorhabditis elegans strains with mutated slicer residues in the endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the mutation in ALG-1 and ALG-2 catalytic residues affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the slicer residues of ALG-1 and ALG-2 contribute differentially to regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the catalytic tetrad of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicer residues of miRNA-specific Argonautes contribute to maintaining levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

Pal S et al. (2017) Curr Biol "CCM-3 Promotes C.elegans Germline Development by Regulating Vesicle Trafficking Cytokinesis and Polarity."

Yu B, Lant B, Derry WB, Krieger JR, Moran MF, Pal S, Tong J, Tian R, Gingras AC

[Curr Biol, 2017]

Cerebral cavernous malformations (CCMs) are vascular defects of the CNS that arise from loss of integrity of the endothelial cells lining blood capillaries, causing leakage of blood into the brain [1]. This results in headaches, seizures, and/or hemorrhagic stroke, depending on the location of the lesion. CCM affects 0.5% of the population and follows anautosomal dominant inheritance pattern caused bymutations in one of the three genes: CCM1 (gene name KRIT1), CCM2 (also known as malcavernin orOSM), and CCM3 (gene name PDCD10) [2, 3], with the earliest onset and most severe prognosis occurring in CCM3 patients [4]. The three CCM genes encode structurally distinct scaffold proteins that function in multiple complexes [5-9]. Using the C.elegans germline as a model of multicellular tube development, we show here that CCM-3 is enriched at the luminal membrane of the germline and the contractile ring of dividing cells in the embryo. Loss of ccm-3 results in defective RAB-11-mediated endocytic recycling, which in turn is necessary for gonadal lumen (rachis) formation, completion of cytokinesis, and localization of cell-surface receptors. CCM-3-mediated localization of anillin and non-muscle myosin to the lateral surfaces of germ cells is required for proper cytoskeletal organization, subsequent oocyte growth, and localization of polarity proteins. Biochemical analysis reveals conservation of the STRIPAK complex and distinct roles for GCK-1 (germinal center kinase III family protein) and striatin/CASH-1 in controlling the localization and function of CCM-3. Taken together, our data establish CCM-3 as a novel regulator of rachis lumenization and polarity establishment during embryogenesis.

Pal, Swati et al. (2015) International Worm Meeting "C. elegans CCM-3: A mother's gift to her fledgling that keeps the traffic moving."

PAL, SWATI, Derry, Brent

[International Worm Meeting, 2015]

Cerebral Cavernous malformations (CCMs) are vascular disorders of central nervous system that arise from loss of integrity of blood capillaries causing blood leakage. This leads to variety of symptoms, including headaches, seizures and hemorrhagic stroke. 3 genes causing familial CCM in humans have been identified: CCM1/2/3; CCM3 mutations being most aggressive. Yet the in vivo function of CCM3 is poorly understood. Caenorhabditis elegans contains orthologues of CCM1 (kri-1) and CCM3 (ccm-3). CCM-3 protein is localized along apical membrane of biological tubes (like excretory canal, intestine and germline) and promotes endocytic recycling to maintain membrane integrity (Lant et al., 2015). To investigate the function of CCM-3 in multicellular tube development we have used C. elegans germline as a model.The nematode germ cells undergo incomplete cytokinesis to create openings into a common lumen called the rachis. Proliferation of mitotic stem cells requires GLP-1/Notch. In ccm-3 mutants there are fewer mitotically proliferating cells with greatly reduced GLP-1 levels. RNAi knockdown of sel-9, which regulates recycling of Notch back to the cell surface, rescues the proliferation defects. Ras/MAPK signalling is required for germ cell pachytene exit and rachis formation. Ablation of ccm-3 causes both a reduction in MAPK signalling and collapse of rachis producing undeveloped oocytes. ccm-3 mutants also fail to present vitellogenin receptor RME-2 on oocytes and show defective VIT-2 uptake. Collectively, these results suggest that loss of ccm-3 results in failure of multiple receptors to get trafficked to membrane surfaces, possibly through defects in endocytic recycling. Consistently, we found that CCM-3 colocalizes with the endosome recycling protein RAB-11. Previous work has shown that loss of anillin proteins results in germline defects that are strikingly similar to ccm-3 mutants, suggesting potential interactions between these scaffolding proteins in promoting rachis development. We observed that CCM-3, along with GCK-1, is required for cortical localization of ANI-1 and non-muscle myosin NMY-2. Knockdown of ccm-3 also results in defective mitotic division of the polarized embryo. Based on these results we hypothesize that CCM-3 functions to couple vesicle trafficking and acto-myosin organization to fine-tune proper assembly and function of the germline. Understanding the CCM-3 interactome may help provide general insights into biological tube development and identify non-surgical approaches for treating CCM patients.

Pal S et al. (1997) Proc Natl Acad Sci U S A "Skn-1: evidence for a bipartite recognition helix in DNA binding."

Walker S, Pelczer I, Schmidt D, Mei-Chu Lo, Thurber S, Pal S

[Proc Natl Acad Sci U S A, 1997]

Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early in the development of Caenorhabditis elegans. This transcription factor contains a basic region, but it binds to DNA as a monomer. Because other transcription factors containing basic regions bind as dimers, this finding implied that Skn represents a new DNA recognition motif. It has been proposed that the basic region helix of Skn is stabilized for binding by tertiary contacts to other parts of the protein. We have tested this proposal by carrying out circular dichroism (CD) and NMR experiments on the Skn domain and five truncated proteins. Our results have shown that the basic region of Skn is unstructured in solution and does not contact other parts of the protein; like other basic region peptides, it folds into a helix only upon binding specifically to DNA. However, there is a stably folded helical module in the Skn domain, and one of the helices in this module terminates immediately before the start of the basic region. This pre-organized helix contains a surface rich in basic amino acids, and we propose that this helix contacts the DNA distal to the basic region proper, providing an extra long helical recognition surface which helps to stabilize monomeric binding. Homology between the Skn domain and several basic-region leucine zipper (bZIP) domains raises the possibility that the affinity and perhaps the specificity of DNA binding by bZIP proteins can be modulated by incorporating a stably folded helical segment that contacts the DNA just below the basic region proper.

Pal R et al. (2019) 3 Biotech "Understanding lipidomic basis of iron limitation induced chemosensitization of drug-resistant Mycobacterium tuberculosis."

Kumar P, Singh S, Fatima Z, Hameed S, Pal R

[3 Biotech, 2019]

Under limited micronutrients condition, <i>Mycobacterium tuberculosis</i> (MTB) has to struggle for acquisition of the limited micronutrients available in the host. One such crucial micronutrient that MTB requires for the growth and sustenance is iron. The present study aimed to sequester the iron supply of MTB to control drug resistance in MTB. We found that iron restriction renders hypersensitivity to multidrug-resistant MTB strains against first-line anti-TB drugs. To decipher the effect of iron restriction on possible mechanisms of chemosensitization and altered cellular circuitry governing drug resistance and virulence of MTB, we explored MTB cellular architecture. We could identify non-intact cell envelope, tampered MTB morphology and diminished mycolic acid under iron restricted MDR-MTB cells. Deeper exploration unraveled altered lipidome profile observed through conventional TLC and advanced mass spectrometry-based LC-ESI-MS techniques. Lipidome analysis not only depicted profound alterations of various lipid classes which are crucial for pathogenecity but also exposed leads such as indispensability of iron to sustain metabolic, genotoxic and oxidative stresses. Furthermore, iron deprivation led to inhibited biofilm formation and capacity of MTB to adhere buccal epithelial cells. Lastly, we demonstrated enhanced survival of <i>Mycobacterium-</i>infected <i>Caenorhabditis elegans</i> model under iron limitation. The present study offers evidence and proposes alteration of lipidome profile and affected virulence traits upon iron chelation. Taken together, iron deprivation could be a potential strategy to rescue MDR and enhance the effectiveness of existing anti-TB drugs.

PAL, SWATI et al. (2013) International Worm Meeting "Deletion of ccm-3 in C. elegans promotes increased accumulation of reactive oxygen species resulting in apoptosis of germline cells."

Derry, W. Brent, Yu, Bin, PAL, SWATI

[International Worm Meeting, 2013]

Cerebral Cavernous malformations (CCMs) are the disorders of capillaries in the central nervous system that result from a loss of integrity in endothelial cells. CCMs affect approximately 1 in 500 individuals and presents in patients a variety of pathophysiological symptoms that range from mild headaches to severe hemorrhagic stroke. Till date, mutations in three genes have been identified that account for approximately 90% of patients with familial disease, ccm-1, ccm-2 and ccm-3. The most severe prognosis is associated with ccm-3 mutations, but the CCM3 signalling pathway has not been resolved. In contrast to the familial form, sporadic CCMs have not been linked with any genetic loci. The varying degrees of symptoms and severity of disease in patients with the same mutation in either of the familial CCM genes suggests the existence of modifier genes. We have shown that ccm-3 (or C14A4.11) affects multiple tissues in C. elegans. For example, ccm-3 homozygote mutants have truncated excretory canals as well as defective oocyte and spermatocyte development that renders them sterile. The oocytes do not grow to the proper size and undergo massive waves of apoptosis, which can be rescued by expression of wild-type ccm-3 gene. Because mammalian CCM genes have been shown to affect reactive oxygen species (ROS), we cultivated ccm-3 mutants in presence of N-acetyl cysteine (NAC), a ROS scavenger, and observed partial rescue of the germline phenotype. NAC also partially protected germlines of wild-type animals from radiation-induced apoptosis. Because the Ras/MAPK pathway has been shown to promote apoptosis in the germline, we are now examining the status of activated MPK-1/ERK in ccm-3 mutant germlines. CCM-3 physically interacts with the germinal center kinase III (GCK-III) family of proteins to negatively regulate ERK activation and apoptosis in mammals. Ablation of the sole GCK-III gene in the worm, gck-1, also results in defective oocyte development that resembles ccm-3 mutants. We hypothesize that CCM-3 and its binding partner GCK-1 are required for proper growth and survival of oocytes.