Biomedical subjects

Lawrence Lum

Publications and source records attributed to Lawrence Lum.

10 recordsLinked to original sources

PubMed2006

Prevalence of off-target effects in Drosophila RNA interference screens.

RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

Animals↗

PubMed2006

A genomewide screen for components of the RNAi pathway in Drosophila cultured cells.

Posttranscriptional silencing by RNAi is initiated by dsRNAs that are processed into siRNAs that ultimately target homologous mRNAs for degradation. We used luciferase reporter constructs and a cultured cell-based assay to perform a genomewide screen for components of the RNAi pathway in Drosophila melanogaster. The screen identified seven genes that affect the RNAi response, five with previously described function (AGO2, Tis11, Hsc70-3, Hsc70-4, and hdc) and two annotated genes (CG17265 and CG10883).

Animals↗

PubMed2006

The ihog cell-surface proteins bind Hedgehog and mediate pathway activation.

The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown here to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response.

Animals↗

PubMed2006

Minimizing the risk of reporting false positives in large-scale RNAi screens.

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

Databases, Genetic↗

PubMed2004

Extensive phosphorylation of Smoothened in Hedgehog pathway activation.

The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in Drosophila have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes in vivo and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1alpha in Hh pathway activation.

Amino Acid Sequence↗

PubMed2004

The Hedgehog response network: sensors, switches, and routers.

The Hedgehog (Hh) signaling pathway is intimately linked to cell growth and differentiation, with normal roles in embryonic pattern formation and adult tissue homeostasis and pathological roles in tumor initiation and growth. Recent advances in our understanding of Hh response have resulted from the identification of new pathway components and new mechanisms of action for old pathway components. The most striking new finding is that signal transmission from membrane to cytoplasm proceeds through recruitment, by the seven-transmembrane protein Smoothened, of an atypical kinesin, which routes pathway activation by interaction with other components of a complex that includes the latent zinc finger transcription factor, Ci.

Animals↗

PubMed2003

Evidence for a critical role of the tumor necrosis factor alpha convertase (TACE) in ectodomain shedding of the p75 neurotrophin receptor (p75NTR).

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.

ADAM Proteins↗

PubMed2003

Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells.

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

Animals↗

PubMed2003

Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2.

The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Here, we present evidence that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo.

Amino Acid Sequence↗

PubMed2003

Potential role for ADAM15 in pathological neovascularization in mice.

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.

ADAM Proteins↗