Jenn‐Yah Yu

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 beta-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.

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) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.