MicroRNA maturation: stepwise processing and subcellular localization

Abstract

MicroRNAs (miRNAs) constitute a novel, phylogenetically extensive family of small RNAs (~22 nucleotides) with potential roles in gene regulation. Apart from the ®nding that miRNAs are produced by Dicer from the precursors of~70 nucleotides (pre-miRNAs), little is known about miRNA biogenesis. Some miRNA genes have been found in close conjunction, suggesting that they are expressed as single transcriptional units.Here, we present in vivo and in vitro evidence that these clustered miRNAs are expressed polycistronically and are processed through at least two sequential steps: (i) generation of the~70 nucleotide pre-miRNAs from the longer transcripts (termed pri-miRNAs); and (ii) processing of pre-miRNAs into mature miRNAs. Subcellular localization studies showed that the ®rst and second steps are compartmentalized into the nucleus and cytoplasm, respectively, and that the pre-miRNA serves as the substrate for nuclear export. Our study suggests that the regulation of miRNA expression may occur at multiple levels, including the two processing steps and the nuclear export step. These data will provide a framework for further studies on miRNA biogenesis.

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

“…Furthermore, similar to the miRNA processing Dicer-like protein in plants, DrnB is present in the nucleus [73] where it seems to be responsible for both generating pre-miRNA and for maturation of the mir-5p and −3p. In contrast, animals carry Drosha, which acts in the nucleus where it cleaves pri-miRNA to pre-miRNA, which is further processed by cytoplasmic Dicer to miRNA-duplexes [10,74]. No obvious homolog to Drosha is present in D. discoideum eventhough the domain structure of DrnB is similar[30].…”

Evolutionary patterns of non-coding RNAs

“…Many of the known microRNAs appear in clusters on a single poly-cistronic transcript [239,294,220,221]. The mir-17 family, for instance, consists of numerous paralogs of three apparently non-homologous sequences.…”

“…MicroRNAs are processed from long primary precursors (pre-miRNAs) [239,238]. Unlike the majority of snoRNAs, neither the genomic location of miRNAs coincides with a specific genomic context, nor is their transcription performed by a single typical mechanism.…”

“…This process allows folding of the premiRNA such that the miRNA precursor (pre-miRNA) forms a stem-loop structure, which in turn is cleaved by nuclear RNase III Drosha [107,46,239,238]. Both, the mature miRNAs and the pre-miRNA can undergo A to I RNA editing by an RNA-specific adenosine deaminase (ADAR) [257].…”

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

“…The necessity to evaluate the proteome is based on the knowledge that higher organisms have several means for controlling cellular function and that proteins mediate the greater part of biological events in the cell, even though certain RNAs can act as effector molecules. However, there are several factors that prevent the linear association of RNA and protein levels (transcription to translation) 15,16 , including single genes that can be translated in several protein products, differential responsivity of mRNA and proteins to intra-and extracellular stimuli, post-transcriptional changes in mRNA (alternative splicing, RNA editing), post-translational modifications of proteins that affect functionality and trafficking (phosphorylation, glycosylation, methylation, palmitoylation) 17 and, most recently, the discovery of microRNAs, which regulate the conversion of mRNAs to proteins [18][19][20] . To evaluate comprehensively the cellular alterations in health and disease, it is essential to determine the alternations in proteins in as complete a manner as possible 21 .…”