Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific ␤-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy. RNA interference (RNAi) has become a powerful and widely used tool for the analysis of gene function in invertebrates and plants (reviewed in ref. 1). Introduction of double-stranded RNA (dsRNA) into the cells of these organisms leads to the sequence-specific destruction of endogenous RNAs that match the dsRNA. During RNAi, long dsRNA molecules are processed into 19-to 23-nt RNAs known as short-interfering RNAs (siRNAs) that serve as guides for enzymatic cleavage of complementary RNAs (2-10). In addition, siRNAs can function as primers for an RNA-dependent RNA polymerase that synthesizes additional dsRNA, which in turn is processed into siRNAs, amplifying the effects of the original siRNAs (11,12). Although the overall process of siRNA inhibition has been characterized, the specific enzymes that mediate siRNA function remain to be identified.In mammalian cells, dsRNA is processed into siRNAs (13-16), but RNAi with dsRNA has not been successful in most cell types because of nonspecific responses elicited by dsRNA molecules longer than about 30 nt (17). However, Tuschl and coworkers (13, 18) recently made the remarkable observation that transfection of synthetic 21-nt siRNA duplexes into mammalian cells effectively inhibits endogenous genes in a sequence-specific manner. These siRNA duplexes are too short to trigger the nonspecific dsRNA responses, but they still cause destruction of complementary RNA sequences (19). It is not known whether siRNAs in mammalian cells also prime synthesis of dsRNA to form additional siRNAs. The recent discovery of large numbers of microRNA genes (reviewed in ref. 20) raises the prospect that the cellular machinery necessary for siRNA inhibition in mammalian cells may be linked to normal processes of gene regulation.In the hope of applying...

We have developed an in situ hybridization procedure for the detection of microRNAs (miRNAs) in tissue sections from mouse embryos and adult organs. The method uses highly specific washing conditions for RNA oligonucleotide probes conjugated to a fluorescein hapten. We show that this method detects predominantly mature miRNAs rather than the miRNA precursors or primary transcripts. We have determined expression patterns for several miRNAs expressed in the developing and adult nervous system, including miR-124a, miR-9, miR-92, and miR-204. Whereas miR-124a is expressed in neurons, miR-9 is expressed in neural progenitors and some neurons, and miR-204 is expressed in the choroid plexus, retinal pigment epithelium, and ciliary body. miR-204 is located in an intron of the TRPM3 gene, and the TRPM3 mRNA is coexpressed with miR-204 in the choroid plexus. We also find that primary transcripts for miR-124a and miR-9 genes are expressed in patterns similar to their respective mature miRNAs. The ability to visualize expression of specific miRNAs in embryos and tissues should aid studies on miRNA function.

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5' base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.

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