ABSTRACT
MicroRNAs (miRNAs) constitute a novel class of short, non-coding RNAs that play crucial roles as silencers of gene expression during biological processes such as development, growth control, cell death, and differentiation. To ensure correct function, the expression of miRNAs must be tightly regulated, a process that is believed to take place at the promoter level. Regulation of the miRNA processing cascade has only recently been shown to play an important role as well and we envision the discovery of additional factors affecting or modulating miRNA processing and ultimately miRNA expression. The biochemical analysis of such factors requires a robust assay to monitor miRNA processing. Here, we discuss protocols and techniques that were used to investigate how the expression of brain-specific miRNA miR-138 is controlled at the level of precursor-miRNA (pre-miRNA) processing.
Subject(s)
MicroRNAs/genetics , MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , Ribonuclease III/metabolism , Base Sequence , Cytoplasm/metabolism , DNA-Directed RNA Polymerases/metabolism , HeLa Cells , Humans , In Vitro Techniques , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Viral Proteins/metabolismABSTRACT
microRNAs (miRNAs) are endogenous, noncoding approximately 22-nucleotide RNA molecules that have recently emerged as fundamental, post-transcriptional regulators of cognate target gene expression. Many mammalian miRNAs are expressed in a tissue-specific manner, a phenomenon that has so far been attributed to transcriptional regulation. We here show by Northern blots and in situ hybridization experiments that the expression of mammalian miRNAs can be regulated at the post-transcriptional level. In particular, miR-138 is spatially restricted to distinct cell types, while its precursor, pre-miR-138-2, is ubiquitously expressed throughout all tissues analyzed. Furthermore, pre-miR-138-2 is exported from the nucleus to the cytoplasm, suggesting that cleavage of this pre-miRNA by Dicer is restricted to certain tissues and cell types. Thus, differential processing of pre-miRNAs might be an alternative mechanism to control miRNA function.
Subject(s)
MicroRNAs/genetics , RNA Processing, Post-Transcriptional , Animals , Base Sequence , Cell Line , Gene Expression Regulation , HeLa Cells , Humans , Mice , Molecular Sequence Data , RNA Precursors/genetics , Restriction MappingABSTRACT
A crucial step in the RNA interference (RNAi) pathway involves the assembly of RISC, the RNA-induced silencing complex. RISC initially recognizes a double-stranded short interfering RNA (siRNA), but only one strand is finally retained in the functional ribonucleoprotein complex. The non-incorporated strand, or 'passenger' strand, is removed during the assembly process and most probably degraded thereafter. In this report, we show that the passenger strand is cleaved during the course of RISC assembly following the same rules established for the siRNA-guided cleavage of a target RNA. Chemical modifications impairing the cleavage of the passenger strand also impair the cleavage of a target RNA in vitro as well as the silencing of a reporter gene in vivo, suggesting that passenger strand removal is facilitated by its cleavage during RISC assembly. Interestingly, target RNA cleavage can be rescued if an otherwise non-cleavable passenger strand shows a nick at the scissile phosphodiester bond, which further indicates that the cleavage event per se is not essential.
Subject(s)
Gene Silencing , RNA Interference , RNA, Small Interfering/metabolism , RNA/metabolism , Genes, Reporter , HeLa Cells , Humans , Multiprotein Complexes , RNA/genetics , RNA, Small Interfering/geneticsABSTRACT
In Drosophila, Dicer-2 requires the double-stranded RNA binding protein R2D2, to mediate the assembly of short interfering RNAs into the RNA-induced silencing complex. New data show that Dicer-1 also requires a double-stranded RNA binding protein called Loquacious for efficient microRNA-mediated gene silencing.
