Young Sik Lee

Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways

The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila.

A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 holo-RISC complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.

Making a better RNAi vector for Drosophila: Use of intron spacers

Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi). This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster. Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method. We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing. A single copy of the transgene effectively and uniformly silences expression of a target gene (white) in transgenic flies. We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene. The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner. Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure.

Silencing by small RNAs is linked to endosomal trafficking.

Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

Chibby, a nuclear β-catenin-associated antagonist of the Wnt/Wingless pathway

Inappropriate activation of downstream target genes by the oncoprotein β-catenin is implicated in development of numerous human cancers. β-catenin and its fruitfly counterpart Armadillo act as a coactivator in the canonical Wnt/Wingless pathway by binding to Tcf/Lef transcription factors. Here we report a conserved nuclear protein, named Chibby, which was identified in a screen for proteins that directly interact with the C-terminal region of β-catenin. In mammalian cultured cells we demonstrate that Chibby inhibits β-catenin-mediated transcriptional activation by competing with Lef-1 to bind to β-catenin. Inhibition of Drosophila Chibby by RNA interference results in segment polarity defects that mimick a wingless gain-of-function phenotype, and overexpression of the wingless target genes engrailed and Ultrabithorax. In addition, epistasis experiments indicate that chibby acts downstream of wingless and upstream of armadillo.

Identification and characterization of new microRNAs from pig

MicroRNAs (miRNAs) are small regulatory RNAs that direct the posttranscriptional repression of cognate messenger RNAs. Despite increasing evidence for diverse roles of miRNAs in biological processes, little is known about miRNAs in pig. We describe the first experimental identification of porcine miRNAs by sequence analysis of a cDNA library of small RNAs from porcine fibroblast cells. We identified 25 distinct porcine miRNAs, of which 19 are previously unreported, and define 14 new miRNA families in pig. Most of the cloned miRNAs are expressed ubiquitously in all porcine tissues examined, whereas some miRNAs are expressed preferentially in specific tissues. Our results enrich the porcine miRNA database and provide useful information for investigating biological functions of miRNAs in pig.

Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.

The endogenous siRNA pathway in Drosophila impacts stress resistance and lifespan by regulating metabolic homeostasis

Small non‐coding RNAs regulate gene expression in a sequence‐specific manner. In Drosophila, Dicer‐2 (Dcr‐2) functions in the biogenesis of endogenous small interfering RNAs (endo‐siRNAs). We identified 21 distinct proteins that exhibited a ⩾1.5‐fold change as a consequence of loss of dcr‐2 function. Most of these were metabolic genes implicated in stress resistance and aging. dcr‐2 Mutants had reduced lifespan and were hypersensitive to oxidative, endoplasmic reticulum, starvation, and cold stresses. Furthermore, loss of dcr‐2 function led to abnormal lipid and carbohydrate metabolism. Our results suggest roles for the endo‐siRNA pathway in metabolic regulation and defense against stress and aging in Drosophila.

Methionine sulfoxide reductase B in the endoplasmic reticulum is critical for stress resistance and aging in Drosophila.

Methionine sulfoxide reductase B (MsrB) is an enzyme that repairs oxidatively damaged proteins by specifically reducing methionine-R-sulfoxide back to methionine. Three MsrBs, localized in different cellular compartments, are expressed in mammals. However, the physiological roles of each MsrB with regard to its location remain poorly understood. Here, we expressed endoplasmic reticulum (ER)-targeted human MsrB3A (hMsrB3A) in Drosophila and examined its effects on various phenotypes. In two independent transgenic lines, both ubiquitous and neuronal expression of hMsrB3A rendered flies resistant to oxidative stress. Interestingly, these flies also showed significantly enhanced cold and heat tolerance. More strikingly, expression of hMsrB3A in the whole body and nervous system extended the lifespan of fruit flies at 29 °C by 43-50% and 12-37%, respectively, suggesting that the targeted expression of MsrB in the ER regulates Drosophila lifespan. A significant increase in lifespan was also observed at 25 °C only when hMsrB3A was expressed in neurons. Additionally, hMsrB3A overexpression significantly delayed the age-related decline in locomotor activity and fecundity. Taken together, our data provide evidence that the ER type of MsrB, MsrB3A, plays an important role in protection mechanisms against oxidative, cold and heat stresses and, moreover, in the regulation of fruit fly aging.

The Role of α-Amino Group of the N-terminal Serine of β Subunit for Enzyme Catalysis and Autoproteolytic Activation of Glutaryl 7-Aminocephalosporanic Acid Acylase

Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (αβ)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free α-amino group of the N-terminal Ser-199 of the β subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pK a = 6.0, which could be attributed to the α-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the α-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own α-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.