MicroRNAs in the Pineal Gland : miR-483 REGULATES MELATONIN SYNTHESIS BY TARGETING ARYLALKYLAMINE N-ACETYLTRANSFERASE

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.

To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.

The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.