RNA interference: genetic wand and genetic watchdog.

In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. The apparently widespread nature of RNAi in eukaryotes, ranging from trypanosome to mouse, has sparked great interest from both applied and fundamental standpoints. Here we review the technical improvements being made to increase the experimental potential of this technique. We also discuss recent advances in uncovering the proteins that act during the RNAi process, discoveries that have revealed enticing links between transposition, transgene silencing and RNAi.

LET-413 is a basolateral protein required for the assembly of adherens junctions in Caenorhabditis elegans.

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.

A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma.

The proto-oncogene c-erbB-2 is overexpressed in 25-30% of breast cancers through increased transcription and amplification of the gene. We have previously described a factor, OB2-1 which upregulates c-erbB-2 transcription and which is closely related to the developmentally regulated transcription factor, AP-2. Further analysis of affinity purified OB2-1 has now shown that it is in fact a combination of proteins from three AP-2-related genes, the previously described AP-2alpha gene and two new human family members, AP-2beta and AP-2gamma whose cloning and characterisation are described here. All three AP-2 proteins show a high degree of homology and are capable of binding to the c-erbB-2 promoter as homo- or heterodimers. The three proteins can also activate a c-erbB-2 reporter construct, but AP-2alpha and AP-2gamma are 3-4 times more active in this regard than AP-2beta. In addition both AP-2alpha and AP-2gamma are expressed at elevated levels in the majority of c-erbB-2 overexpressing mammary tumour lines examined. Mechanisms which may have led to the increased AP-2 levels in these cells are discussed.

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles.

lir-2, lir-1 and lin-26 encode a new class of zinc-finger proteins and are organized in two overlapping operons both in Caenorhabditis elegans and in Caenorhabditis briggsae.

lin-26, which encodes a unique Zn-finger protein, is required for differentiation of nonneuronal ectodermal cells in Caenorhabditis elegans. Here, we show that the two genes located immediately upstream of lin-26 encode LIN-26-like Zn-finger proteins; hence their names are lir-1 and lir-2 (lin-26 related). lir-2, lir-1, and lin-26 generate several isoforms by alternative splicing and/or trans-splicing at different positions. On the basis of their trans-splicing pattern, their intergenic distances, and their expression, we suggest that lir-2, lir-1, and lin-26 form two overlapping transcriptional operons. The first operon, which is expressed in virtually all cells, includes lir-2 and long lir-1 isoforms. The second operon, which is expressed in the nonneuronal ectoderm, includes short lir-1 isoforms, starting at exon 2 and lin-26. This unusual genomic organization has been conserved in C. briggsae, as shown by cloning the C. briggsae lir-2, lir-1, and lin-26 homologs. Particularly striking is the sequence conservation throughout the first lir-1 intron, which is very long in both species. Structural conservation is functionally meaningful as C. briggsae lin-26 is also expressed in the nonneuronal ectoderm and can complement a C. elegans lin-26 null mutation.

The developmentally regulated transcription factor AP-2 is involved in c-erbB-2 overexpression in human mammary carcinoma.

Overexpression of the c-erbB-2/HER2 protooncogene in breast carcinoma is controlled not only by the degree of amplification of the gene but also at the level of gene transcription. Thus, whether or not the gene is amplified, the activity of the c-erbB-2 promoter is enhanced in overexpressing cells through the binding of an additional transcription factor, OB2-1, whose activity is increased in these lines. Here we describe further characterization of OB2-1 and show that it is identical to the developmentally regulated transcription factor AP-2. Functional assays confirm that AP-2 is able to regulate c-erbB-2 expression in mammary-derived cell lines. Furthermore, although AP-2 is barely detectable in cells with the low c-erbB-2 expression phenotype, protein levels are clearly elevated in a panel of c-erbB-2-overexpressing lines. These findings demonstrate an important role for this transcription factor in human cancer.

Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors.

The AP-2 transcription factor has been shown to play an important role in the development of tissues of ectodermal origin and has also been implicated in mammary oncogenesis. It has recently been found that AP-2 is encoded by a family of related genes, AP-2alpha, AP-2beta, and AP-2gamma. As a further step in understanding the role each of these genes has in development, we have used fluorescence in situ hybridization to map the chromosomal locations of the mouse and human homologues of the newly isolated AP-2beta and AP-2gamma genes. Tcfap2b and Tcfap2c map to mouse chromosomes 1A2-4 and 2H3-4, respectively, while TFAP2B and TFAP2C map to human chromosomes 6p12 and 20q13.2, the latter being a region that is frequently amplified in breast carcinoma.