Small RNAs beyond Model Organisms: Have We Only Scratched the Surface?

Small RNAs (sRNAs) are essential regulators in the adaptation of bacteria to environmental changes and act by binding targeted mRNAs through base complementarity. Approximately 550 distinct families of sRNAs have been identified since their initial characterization in the 1980s, accelerated by the emergence of RNA-sequencing. Small RNAs are found in a wide range of bacterial phyla, but they are more prominent in highly researched model organisms compared to the rest of the sequenced bacteria. Indeed, Escherichia coli and Salmonella enterica contain the highest number of sRNAs, with 98 and 118, respectively, with Enterobacteriaceae encoding 145 distinct sRNAs, while other bacteria families have only seven sRNAs on average. Although the past years brought major advances in research on sRNAs, we have perhaps only scratched the surface, even more so considering RNA annotations trail behind gene annotations. A distinctive trend can be observed for genes, whereby their number increases with genome size, but this is not observable for RNAs, although they would be expected to follow the same trend. In this perspective, we aimed at establishing a more accurate representation of the occurrence of sRNAs in bacteria, emphasizing the potential for novel sRNA discoveries.

Glucan particles for selective delivery of siRNA to phagocytic cells in mice.

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 µm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes.

The lipid droplet-associated fat specific protein 27 (FSP27) suppresses lipolysis and thereby enhances triglyceride accumulation in adipocytes. We and others have recently found FSP27 to be a remarkably short-lived protein (half-life, 15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, we tested the hypothesis that lipolytic agents such as tumor necrosis factor-α (TNF-α) and isoproterenol modulate FSP27 levels to regulate FFA release. Consistent with this concept, we showed that the lipolytic actions of TNF-α, interleukin-1ß (IL-1ß), and IFN-γ are accompanied by marked decreases in FSP27 expression and lipid droplet size in mouse adipocytes. Similar depletion of FSP27 using short interfering RNA (siRNA) mimicked the lipolysis-enhancing effect of TNF-α, while maintaining stable FSP27 levels using expression of hemagglutinin epitope-tagged FSP27 blocked TNF-α-mediated lipolysis. In contrast, we show the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 levels and a delayed degradation rate corresponding to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism for restraining excessive lipolysis by catecholamines, is mimicked by forskolin or 8-bromo-cAMP treatment and is prevented by the protein kinase A (PKA) inhibitor KT5720 or by PKA depletion using siRNA. Taken together, these data identify the regulation of FSP27 as an important intermediate in the mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol.

Identification of the lipid droplet targeting domain of the Cidea protein.

Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domains. Expression of full-length Cidea in undifferentiated 3T3-L1 cells or COS-1 cells increases total cellular triglyceride and strikingly alters the morphology of lipid droplets by enhancing their size and reducing their number. Remarkably, both lipid droplet binding and increased triglyceride accumulation are also elicited by expression of only the carboxy-terminal 104 amino acids, indicating this small domain directs lipid droplet targeting and triglyceride shielding. However, unlike the full-length protein, expression of the carboxy-terminus causes clustering of small lipid droplets but not the formation of large droplets, identifying a novel function of the N terminus. Furthermore, human Cidea promotes lipid storage via lipolysis inhibition, as the expression of human Cidea in fully differentiated 3T3-L1 adipocytes causes a significant decrease in basal glycerol release. Taken together, these data indicate that the carboxy-terminal domain of Cidea directs lipid droplet targeting, lipid droplet clustering, and triglyceride accumulation, whereas the amino terminal domain is required for Cidea-mediated development of enlarged lipid droplets.

Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium.

Hammerhead ribozymes are one of the most studied classes of ribozymes so far, from both the structural and biochemical point of views. The activity of most hammerhead ribozymes is cation-dependent. Mg2+ is one of the most abundant divalent cations in the cell and therefore plays a major role in cleavage activity for most hammerhead ribozymes. Besides Mg2+, cleavage can also occur in the presence of other cations such as Mn2+. The catalytic core of hammerhead ribozymes is highly conserved, which could contribute to a preference of hammerhead ribozymes toward certain cations. Here, we show a naturally occurring variation in the catalytic core of hammerhead ribozymes, A6C, that can favor one metallic ion, Mn2+, over several other cations.

Association of a homozygous nonsense caveolin-1 mutation with Berardinelli-Seip congenital lipodystrophy.

CONTEXT: Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue, resulting in severe dyslipidemia and insulin resistance. In most reported cases, BSCL is due to alterations in either seipin, of unknown function, or 1-acylglycerol-3-phosphate acyltransferase-beta (AGPAT2), which catalyzes the formation of phosphatidic acid. OBJECTIVE: We sought to determine the genetic origin of the unexplained cases of BSCL. We thus sequenced CAV1, encoding caveolin-1, as a candidate gene involved in insulin signaling and lipid homeostasis. CAV1 is a key structural component of plasma membrane caveolae, and Cav1-deficient mice display progressive loss of adipose tissue and insulin resistance. DESIGN: We undertook phenotyping studies and molecular screening of CAV1 in four patients with BSCL with no mutation in the genes encoding either seipin or AGPAT2. RESULTS: A homozygous nonsense mutation (p.Glu38X) was identified in CAV1 in a patient with BSCL born from a consanguineous union. This mutation affects both the alpha- and beta-CAV1 isoforms and ablates CAV1 expression in skin fibroblasts. Detailed magnetic resonance imaging of the proband confirmed near total absence of both sc and visceral adipose tissue, with only vestigial amounts in the dorsal sc regions. In keeping with the lack of adipose tissue, the proband was also severely insulin resistant and dyslipidemic. In addition, the proband had mild hypocalcemia likely due to vitamin D resistance. CONCLUSIONS: These findings identify CAV1 as a new BSCL-related gene and support a critical role for caveolins in human adipocyte function.

Higher adiponectin levels in patients with Berardinelli-Seip congenital lipodystrophy due to seipin as compared with 1-acylglycerol-3-phosphate-o-acyltransferase-2 deficiency.

CONTEXT: Human lipodystrophies are characterized by loss of adipose tissue, insulin resistance, and metabolic complications. The mechanisms linking fat loss to severe insulin resistance remain unclear. Adipokines may have important roles as intermediary players in metabolism. OBJECTIVE: We sought to determine the plasma concentrations of leptin and adiponectin in patients with Berardinelli-Seip congenital lipodystrophy (BSCL) harboring mutations in the genes encoding either 1-acylglycerol-3-phosphate-O-acyltransferase-2 (AGPAT2) or BSCL2/seipin, in comparison with patients with other forms of inherited or acquired lipodystrophies or insulin receptor alterations. DESIGN: Leptin and total and high-molecular-weight adiponectin were measured in plasma of 16 BSCL1/AGPAT2 and 19 BSCL2/seipin patients and compared with heterozygous (n = 22) or nonmutated relatives (controls, n = 30); patients with Dunnigan-type partial lipodystrophy due to lamin A/C mutations (n = 23), HIV-related lipodystrophy (n = 124), and insulin receptor dysfunctions caused by mutations or autoantibodies (n = 17). RESULTS: Leptin was dramatically decreased in BSCL patients as compared with other subgroups. Adiponectin was decreased in BSCL as compared with controls and patients with altered insulin receptor but was discrepant between the two BSCL subgroups. Whereas total and high-molecular-weight adiponectin levels were almost undetectable in BSCL1/AGPAT2 patients, higher levels were detected in BSCL2/seipin patients, comparable with those of patients with partial lipodystrophy. Adiponectin greater than 1.6 mg/liter had a 100% negative predictive value for AGPAT2 mutations in inherited lipodystrophies. CONCLUSIONS: The presence of circulating adiponectin in BSCL2/seipin patients with near absence of adipose tissue outlines the complexity of adiponectin biology. Use of circulating adiponectin might be helpful to guide the genetic investigations in BSCL.

Seipin deficiency alters fatty acid Delta9 desaturation and lipid droplet formation in Berardinelli-Seip congenital lipodystrophy.

Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue and severe insulin resistance. In most cases, BSCL is due to loss-of-function mutations in the genes encoding either seipin of unknown function or 1-acyl-glycerol-3-phosphate O-acyltransferase 2 (AGPAT2) which catalyses the formation of phosphatidic acid from lysophosphatidic acid. We studied the lipid profile of lymphoblastoid cell-lines from 20 BSCL patients with null mutations in the genes encoding either seipin (n=12) or AGPAT2 (n=8) in comparison to nine control cell-lines. In seipin deficient cells, we observed alterations in the pattern of lipid droplets which were decreased in size and increased in number as compared to control cells. We also observed alterations in the triglycerides content as well as in the fatty acid composition from triglycerides and phosphatidylethanolamine, with an increased proportion of saturated fatty acids at the expense of the corresponding monounsaturated fatty acids, reflecting a defect in Delta9-desaturase activity. In AGPAT2 deficient cells, no specific alterations in lipid droplet pattern nor in fatty acid composition was observed but the cellular level of lysophosphatidic acid was increased as compared to that of control and seipin deficient cells. These results indicate that seipin like AGPAT2 is involved in lipid metabolism but exerts a different function. Seipin intervenes at a proximal step in triglycerides and phospholipids biosynthesis being involved in the pathway that links fatty acid Delta9 desaturation to lipid droplet formation. These findings provide new insights into how seipin deficiency causes severe lipodystrophy.