In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes.This system is useful to study regulation of Drosha activity in physiological and pathological conditions.     

Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28

The developmentally regulated RNA-binding protein Lin28 blocks processing of let-7 family microRNAs (miRNAs) in embryonic cells. The molecular basis for this selective miRNA processing block is unknown. Here we find that Lin28 selectively binds the terminal loop region of let-7 precursors in vitro and that the loop mediates miRNA processing inhibition in vivo. Additionally, we identify the domains of Lin28 required for this inhibition. These findings establish a regulatory role for the terminal loop of precursors in miRNA maturation and provide insight into the mechanism by which Lin28 negatively regulates let-7 processing.     

A loop‐to‐base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159

The first step in microRNA (miRNA) biogenesis usually involves cleavage at the base of its fold‐back precursor. Here, we describe a non‐canonical processing mechanism for miRNAs miR319 and miR159 in Arabidopsis thaliana. We found that their biogenesis begins with the cleavage of the loop, instead of the usual cut at the base of the stem–loop structure. DICER‐LIKE 1 (DCL1) proceeds then with three additional cuts until the mature miRNA is released. We further show that the conserved upper stem of the miR319 precursor is essential to organize its biogenesis, whereas sequences below the miRNA/miRNA^* region are dispensable. In addition, the bulges present in the fold‐back structure reduce the accumulation of small RNAs other than the miRNA. The biogenesis of miR319 is conserved in the moss Physcomitrella patens, showing that this processing mechanism is ancient. These results provide new insights into the plasticity of small‐RNA pathways.     

Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing

Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.     

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

Background  

DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.