Rajini R. Mudhasani

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19Arf and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19Arf-p53 signaling, and induces senescence in primary cells.

Dicer inactivation in osteoprogenitor cells compromises fetal survival and bone formation, while excision in differentiated osteoblasts increases bone mass in the adult mouse

MicroRNA attenuation of protein translation has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage. A compelling question is the extent to which miR biogenesis is obligatory for bone formation. Here we show conditional deletion of the Dicer enzyme in osteoprogenitors by Col1a1-Cre compromised fetal survival after E14.5. A mechanism was associated with the post-commitment stage of osteoblastogenesis, demonstrated by impaired ECM mineralization and reduced expression of mature osteoblast markers during differentiation of mesenchymal cells of ex vivo deleted Dicer(c/c). In contrast, in vivo excision of Dicer by Osteocalcin-Cre in mature osteoblasts generated a viable mouse with a perinatal phenotype of delayed bone mineralization which was resolved by 1 month. However, a second phenotype of significantly increased bone mass developed by 2 months, which continued up to 8 months in long bones and vertebrae, but not calvariae. Cortical bone width and trabecular thickness in Dicer(Deltaoc/Deltaoc) was twice that of Dicer(c/c) controls. Normal cell and tissue organization was observed. Expression of osteoblast and osteoclast markers demonstrated increased coupled activity of both cell types. We propose that Dicer generated miRs are essential for two periods of bone formation, to promote osteoblast differentiation before birth, and control bone accrual in the adult.

IFITM-2 and IFITM-3 but Not IFITM-1 Restrict Rift Valley Fever Virus

ABSTRACT We show that interferon-induced transmembrane protein 1 (IFITM-1), IFITM-2, and IFITM-3 exhibit a broad spectrum of antiviral activity against several members of the Bunyaviridae family, including Rift Valley fever virus (RVFV), La Crosse virus, Andes virus, and Hantaan virus, all of which can cause severe disease in humans and animals. We found that RVFV was restricted by IFITM-2 and -3 but not by IFITM-1, whereas the remaining viruses were equally restricted by all IFITMs. Indeed, at low doses of alpha interferon (IFN-α), IFITM-2 and -3 mediated more than half of the antiviral activity of IFN-α against RVFV. IFITM-2 and -3 restricted RVFV infection mostly by preventing virus membrane fusion with endosomes, while they had no effect on virion attachment to cells, endocytosis, or viral replication kinetics. We found that large fractions of IFITM-2 and IFITM-3 occupy vesicular compartments that are distinct from the vesicles coated by IFITM-1. In addition, although overexpression of all IFITMs expanded vesicular and acidified compartments within cells, there were marked phenotypic differences among the vesicular compartments occupied by IFITMs. Collectively, our data provide new insights into the possible mechanisms by which the IFITM family members restrict distinct viruses.

Dicer is required for the formation of white but not brown adipose tissue

Dicer, an enzyme involved in microRNA maturation, is required for proper embryo gastrulation and tissue morphogenesis during mammalian development. Using primary cultures of fibroblasts and pre‐adipocytes, we have previously shown that Dicer is essential for early stages of adipogenic cell differentiation. In this study, we have utilized Dicer‐conditional mice to explore a role for Dicer and microRNA biogenesis in the terminal differentiation of adipocytes in vivo and in the formation of white and brown adipose tissue. Deletion of Dicer in differentiated adipocytes in Dicer‐conditional, aP2‐Cre transgenic mice reduced the level of various adipogenic‐associated transcripts and inhibited lipogenesis in white adipocytes, resulting in a severe depletion of white adipose tissue in mice. In contrast, Dicer was not required in vivo for lipogenesis in brown adipose or for brown fat formation. However, Dicer deletion in brown adipose did decrease the expression of genes involved in thermoregulation. The results of our study provide genetic evidence of a role for microRNA molecules in regulating adipogenesis and reveal distinct requirements for Dicer in the formation of white and brown adipose tissue. J. Cell. Physiol. 226: 1399–1406, 2011.

Oxygen sufficiency controls TOP mRNA translation via the TSC-Rheb-mTOR pathway in a 4E-BP-independent manner.

Cells encountering hypoxic stress conserve resources and energy by downregulating the protein synthesis. Here we demonstrate that one mechanism in this response is the translational repression of TOP mRNAs that encode components of the translational apparatus. This mode of regulation involves TSC and Rheb, as knockout of TSC1 or TSC2 or overexpression of Rheb rescued TOP mRNA translation in oxygen-deprived cells. Stress-induced translational repression of these mRNAs closely correlates with the hypophosphorylated state of 4E-BP, a translational repressor. However, a series of 4E-BP loss- and gain-of-function experiments disprove a cause-and-effect relationship between the phosphorylation status of 4E-BP and the translational repression of TOP mRNAs under oxygen or growth factor deprivation. Furthermore, the repressive effect of anoxia is similar to that attained by the very efficient inhibition of mTOR activity by Torin 1, but much more pronounced than raptor or rictor knockout. Likewise, deficiency of raptor or rictor, even though it mildly downregulated basal translation efficiency of TOP mRNAs, failed to suppress the oxygen-mediated translational activation of TOP mRNAs. Finally, co-knockdown of TIA-1 and TIAR, two RNA-binding proteins previously implicated in translational repression of TOP mRNAs in amino acid-starved cells, failed to relieve TOP mRNA translation under other stress conditions. Thus, the nature of the proximal translational regulator of TOP mRNAs remains elusive.

Functional Interaction of the Retinoblastoma and Ini1/Snf5 Tumor Suppressors in Cell Growth and Pituitary Tumorigenesis

The Ini1 subunit of the SWI/SNF chromatin remodeling complex suppresses formation of malignant rhabdoid tumors in humans and mice. Transduction of Ini1 into Ini1-deficient tumor-derived cell lines has indicated that Ini1 arrests cell growth, controls chromosomal ploidy, and suppresses tumorigenesis by regulating components of the retinoblastoma (Rb) signaling pathway. Furthermore, conditional inactivation of Ini1 in mouse fibroblasts alters the expression of various Rb-E2F-regulated genes, indicating that endogenous Ini1 levels may control Rb signaling in cells. We have reported previously that loss of one allele of Ini1 in mouse fibroblasts results only in a 15% to 20% reduction in total Ini1 mRNA levels due to transcriptional compensation by the remaining Ini1 allele. Here, we examine the effects of Ini1 haploinsufficiency on cell growth and immortalization in mouse embryonic fibroblasts. In addition, we examine pituitary tumorigenesis in Rb-Ini1 compound heterozygous mice. Our results reveal that heterozygosity for Ini1 up-regulates cell growth and immortalization and that exogenous Ini1 down-regulates the growth of primary cells in a Rb-dependent manner. Furthermore, loss of Ini1 is redundant with loss of Rb function in the formation of pituitary tumors in Rb heterozygous mice and leads to the formation of large, atypical Rb(+/-) tumor cells lacking adrenocorticotropic hormone expression. These results confirm in vivo the relationship between Rb and Ini1 in tumor suppression and indicate that Ini1 plays a role in maintaining the morphologic and functional differentiation of corticotrophic cells.

SWI/SNF Chromatin Remodeling Enzyme ATPases Promote Cell Proliferation in Normal Mammary Epithelial Cells

The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma‐related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre‐disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF‐10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three‐dimensional rBM culture, although some BRM‐depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells. J. Cell. Physiol. 223:667–678, 2010.

An essential role for Dicer in adipocyte differentiation.

Dicer is a cellular enzyme required for the processing of pre‐miRNA molecules into mature miRNA, and Dicer and miRNA biogenesis have been found to play important roles in a variety of physiologic processes. Recently, reports of alterations in miRNA expression levels in cultured pre‐adipogenic cell lines during differentiation and findings of differences between the miRNA expression signatures of white and brown adipose have suggested that miRNA molecules might regulate adipocyte differentiation and the formation of adipose tissue. However, direct evidence that miRNAs regulate adipogenesis is lacking. To determine if Dicer and mature miRNA govern adipocyte differentiation, we utilized primary cells isolated from mice bearing Dicer‐conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis. Our results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre‐adipocytes. Furthermore, the requirement for Dicer in adipocyte differentiation is not due to miRNA‐mediated alterations in cell proliferation, as deletion of the Ink4a locus and the prevention of premature cellular senescence normally induced in primary cells upon Dicer ablation fails to rescue adipogenic differentiation in fibroblasts and pre‐adipocytes. J. Cell. Biochem. 110: 812–816, 2010.

Reassessment of the Role of TSC, mTORC1 and MicroRNAs in Amino Acids-Meditated Translational Control of TOP mRNAs

TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

High Content Image-Based Screening of a Protease Inhibitor Library Reveals Compounds Broadly Active against Rift Valley Fever Virus and Other Highly Pathogenic RNA Viruses

High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology.